HK1211315B - Intein-modified proteases, their production and industrial applications - Google Patents

Description

Intein-modified protease, preparation method and industrial use thereof

This application claims priority to U.S. Provisional Application No. 61/744,863, filed October 3, 2012, and U.S. Provisional Application No. 61/783,424, filed March 14, 2013, which are both incorporated herein by reference as if fully set forth.

The sequence listing entitled "Sequence Listing" filed electronically with this application was created on October 3, 2012, has a size of 3,246,349 bytes, and is also incorporated herein by reference as if fully set forth.

Technical Field

The present invention relates to an intein-modified protease, a method for preparing an intein-modified protease, a method for preparing a protease and the use of an intein-modified protease.

Background Art

Proteases are enzymes that hydrolyze proteins and polypeptides into smaller peptides and amino acids. Proteases have found widespread industrial application, particularly in fabric care products, detergents, dishwashing liquids, industrial cleaners, solutions for biofilm removal, and animal feed. Proteases are commonly formulated into or added to detergents for use as stain removers in fabric and clothing washing, or in liquid detergents for cleaning dishes and other items. Proteases are also used in animal feed to help them digest dietary protein. Despite these numerous beneficial uses, proteases are extremely difficult to produce because they not only degrade other proteins but may also degrade themselves. For these reasons, only a very small number of specific proteases have found commercial use. Technologies for modulating protease activity, whether during expression, purification, formulation, in the final product, or during industrial, agricultural, consumer, home care, or feed processing, are valuable in promoting the application of proteases and in discovering new proteases with improved properties.

Summary of the Invention

One aspect of the present invention relates to an intein-modified protease. The intein-modified protease comprises a target protease and an intein fused to the target protease. The intein is fused to the target protease in a position such that the intein can control the activity of the target protease. The intein can effect splicing of the intein-modified protease.

One aspect of the present invention relates to an expression cassette. The expression cassette comprises a polynucleotide encoding an intein-modified protease. The intein-modified protease comprises a target protease and an intein fused to the target protease. The intein is fused to the target protease in a position such that the intein controls the activity of the target protease. The intein is capable of effecting splicing of the intein-modified protease.

One aspect of the present invention relates to an expression cassette comprising a polynucleotide having a sequence at least 90% identical to a reference sequence selected from the group consisting of SEQ ID NO: 44 (pAG2209), SEQ ID NO: 45 (pAG2210), SEQ ID NO: 46 (pAG2211), SEQ ID NO: 47 (pAG2212), SEQ ID NO: 48 (pAG2216), SEQ ID NO: 49 (pAG2217), SEQ ID NO: 50 (pAG2218), SEQ ID NO: 51 (pAG2219), SEQ ID NO: 52 (pAG2220), SEQ ID NO: 53 (pAG2221), SEQ ID NO: 54 (pAG2222), and SEQ ID NO: 55 (pAG2223).

One aspect of the present invention relates to an expression cassette comprising a polynucleotide having a sequence at least 90% identical to the reference sequence of SEQ ID NO: 629 (pET22_iSAV_Hwa_S317_nuc) or SEQ ID NO: 630 (P416GALL-Ura).

One aspect of the present invention relates to a host genetically engineered to express an intein-modified protease. The intein-modified protease comprises a target protease and an intein fused to the target protease. The intein is fused to the target protease in a position such that the intein controls the activity of the target protease. The intein is capable of effecting splicing of the intein-modified protease.

One aspect of the present invention relates to a host comprising any one of the intein-modified proteases described herein.

One aspect of the present invention relates to a method for preparing a protease. The method comprises inducing splicing of an intein-modified protease. The intein-modified protease comprises a target protease and an intein fused to the target protease, the intein fused to the target protease being positioned such that the intein can control the activity of the target protease. The intein is capable of effecting splicing of the intein-modified protease.

One aspect of the present invention relates to a method for modulating the activity of a protease. The method comprises preparing the protease by any of the methods described herein.

One aspect of the present invention relates to an animal feed comprising any one of the intein-modified proteases described herein.

One aspect of the present invention relates to a detergent comprising any one of the intein-modified proteases described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments of the present invention described in detail below will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, the accompanying drawings show preferred embodiments. However, it should be understood that the invention is not limited to the precise arrangements and instrumentalities shown. In the drawings:

Figure 1 shows the activity of Q53521 keratinase in T1 seeds of event AB X 2209. Numbers 1 to 12 refer to the TO events generated by construct pAG2209 (SEQ ID NO: 624).

FIG2 shows the protease activity of Savinase secreted from the culture supernatant of Bacillus subtilis.

FIG3A shows the protease activity of pro-Savinase and the Savinase catalytic domain expressed in the periplasm (P) and protoplasm (S) of E. coli.

FIG3B shows Western blot analysis of pro-Savinase and Savinase catalytic domain expression in the periplasm (P) and protoplasm (S) of E. coli.

FIG4A shows the protease activity of Savinase expressed in the cytoplasm of E. coli SOLR cells.

FIG4B shows the effect of cytoplasmic expression of Savinase on the growth of E. coli SOLR cells.

Figure 5 shows the inhibition assay.

Figure 6 shows the induction analysis.

7A-7B show Western blots of intein splicing in pro-Savinase.

Figure 8 shows the splicing of pro-Savinase using modified mVMA:P77Cd and mTth:P77Cd inteins.

FIG9 shows the assembly profile of trans-spliced proteins.

FIG10 shows visualization of iSavinase detergent inhibition of trans-splicing.

FIG11 shows dilution analysis of iSavinase:S317-Gp41-1 NI and IC using detergent-regulated trans-splicing.

FIG12 shows the removal of blood stains using trans-splicing iSavinase.

FIG13 shows the removal of blood, milk, and ink stains using trans-splicing iSavinase.

FIG14 shows detergent stability testing of trans-splicing iSavinase: S317-Gp41-1 NI and IC.

FIG15 shows detergent inhibition analysis of cis-splicing iSavinase constructs.

FIG. 16 shows detergent dilution analysis of cis-splicing iSavinase constructs.

Figure 17 shows temperature induction analysis of selected cis-splicing iSavinase constructs: iproSavinaseS135:15, iproSavinaseS135:145, iproSavinaseS135:153, iproSavinaseS135:155, iproSavinaseS135:155-var7 and control constructs ProSavinase and inactive proSaviH62.

Figures 18A-18C show temperature induction analysis of selected cis-splicing iSavinase constructs: proSavinaseS135:Cth_ATPase_BIL, proSavianseS135:Mja_Klba and control constructs, ProSavinase and inactive proSaviH62.

Figures 19A-19F show detergent inhibition analysis of the cis-splicing iSavinase construct proSavinaseS135:Cth_ATPase_BIL and control constructs proSavinase and inactive proSaviH62 at 20°C and 37°C.

Figures 20A-20D show detergent inhibition analysis at 20°C and 37°C of selected cis-splicing iSavinase constructs: proSavinase, proSaviH62, AS15, and AS48.

DETAILED DESCRIPTION

Certain terms used in the following description are for convenience only and are not limiting. The words "right," "left," "top," and "bottom" indicate directions in the drawings for reference. Unless otherwise specified, the words "a" and "one" used in the claims and corresponding parts of the specification are defined to include one or more of the referenced items. This term includes the words specifically mentioned above, their derivatives, and words of similar meaning. The phrase "at least one" followed by two or more items, such as "A, B, or C," refers to any one of A, B, or C and any combination thereof.

One embodiment includes an intein-modified protease. The intein-modified protease can include a target protease and an intein fused to the target protease, the fusion being positioned so that the intein can control the activity of the target protease. The intein can enable splicing of the intein-modified protease. The intein can be fused to the interior of the target protease, meaning that the intein sequence is inserted into the sequence of the target protease. The intein can be fused to the exterior of the target protease. An externally fused intein can enable trans-splicing, or cis-splicing. An internally fused intein can enable cis-splicing.

The position of the fusion is such that the intein can substantially reduce or inhibit the activity of the target protease. The substantially reduced activity of the target protease can include, compared to the target protease, an activity reduction of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100%, or a percentage in the range between any two numbers. As used herein, "protease" refers to an enzyme or part thereof that catalyzes the hydrolysis of peptide bonds. The enzyme can be, but is not limited to, an amino acid sequence or protein with catalytic peptide bond hydrolysis activity as described herein. The enzyme can be a variant of an amino acid sequence or protein with catalytic peptide bond hydrolysis activity as described herein, wherein the variant is a mutant and/or part of an amino acid sequence or protein. The variant can have at least 40% of the activity of an amino acid sequence or protein with catalytic peptide bond hydrolysis activity.

The target protease may be a peptide hydrolase classified under the EC 3.4 classification. Under this classification, target proteases may include those classified under EC 3.4.99, EC 3.4.21.62, serine proteases, alkaline proteases, keratinase, and other enzymes. Other target proteases that may be part of the intein-modified proteases herein include, but are not limited to, metalloproteases, cysteine proteases, aspartic proteases, and ATP-dependent proteases. The subtilisin family of proteases, Savinase, P29600 (SEQ ID NO: 1), and Keratinase, Q53521 (SEQ ID NOs: 12 and 621), may be target proteases among the intein-modified proteases herein. While Savinase and P29600 may have diverse applications in fabric care and detergent products, they may also find use in animal feed, with Keratinase and Q53521 being useful in feed products. Other target proteases may include a subtilisin from B. lentus (BL, P29599, SEQ ID NO: 2); a subtilisin from B. pumilus (PO7518, SEQ ID NO: 3); a subtilisin from B. subtilis (E, P04189, SEQ ID NO: 4); a subtilisin from B. licheniformis (DY, P00781, SEQ ID NO: 5); a subtilisin from B. amyloliquefaciens (BPN, P00782, SEQ ID NO: 6); a subtilisin from Bacillus sp. strain TA39 (P28842, SEQ ID NO: 7); a subtilisin from Geobacillus stearothermophilus (P28843, SEQ ID NO: 8); a subtilisin from B. licheniformis (DY, P00781, SEQ ID NO: 9); a subtilisin from B. amyloliquefaciens (BPN, P00782, SEQ ID NO: 10); a subtilisin from Bacillus sp. strain TA39 (P28842, SEQ ID NO: 11); a subtilisin from B. stearothermophilus) (J, P29142, SEQ ID NO: 8); subtilisin from B. subtilis subsp. Natto (NAT, P35835, SEQ ID NO: 9); subtilisin from B. licheniformis (Carlsberg, P00780, SEQ ID NO: 10); subtilisin from B. subtilis subsp. Amylosacchariticus (amylosacchariticus, P00780, SEQ ID NO: 11); and acid fungal protease from Trichoderma reesei (SEQ ID NO: 718).

In embodiments, the target protease of the intein-modified protease can comprise, consist essentially of, or consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 (P29600), SEQ ID NO: 2 (P29599), SEQ ID NO: 3 (P07518), SEQ ID NO: 4 (P04189), SEQ ID NO: 5 (P00781), SEQ ID NO: 6 (P00782), SEQ ID NO: 7 (P28842), SEQ ID NO: 8 (P29142), SEQ ID NO: 9 (P35835), SEQ ID NO: 10 (P00780), SEQ ID NO: 11 (P00783), SEQ ID NO: 12 (Q53521), SEQ ID NO: 57 (ProSavinase), SEQ ID NO: 58 (catalytic domain of Savinase), and SEQ ID NO: 13 (Q53522). The reference sequences in the group consisting of ID NO: 718 (acid fungal protease) have at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity.

Determining the percent identity of two amino acid sequences or two nucleic acid sequences can include aligning and comparing the amino acid residues or nucleotides at the corresponding positions of the two sequences. If all positions of the two sequences are occupied by the same amino acid residue or nucleotide, the sequences are said to have 100% identity. Percent identity can be determined by the Smith-Waterman algorithm (Smith TF, Waterman MS 1981 "Identification of Common Molecular Subsequences," J Mol Biol 147:195-197, incorporated herein by reference as if fully set forth).

In one embodiment, the protease can be a target protease with less than 100% identity to the amino acid reference sequence cited, or a variant of the protease with the amino acid reference sequence. In another embodiment, a polynucleotide sequence encoding a protease with less than 100% identity to the protease encoded by the reference sequence can encode a variant of the protease encoded by the reference sequence. The amino acid sequence or variant of the protease can have at least 40% of the activity of the amino acid sequence or protease. The variant of the protease can be a part or fragment of the protease. Its fragment or part can include 100, 150, 200, 300, 400, 600 or more continuous amino acids, for example 700. The function of the protease (which can be a target protease, variant or fragment or its part) can be determined using any known method. The function of the protease can include the ability to hydrolyze peptide bonds. For example, the method of determination is recorded in Examples 7, 14 and 15 of this paper.

In one embodiment, a protease is provided that is at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a protease having a sequence as set forth in any one of SEQ ID NOs: 1-12, 57-58, and 718 (having amino acids 6, 10 to 50, 10 to 100, 10 to 150, 10 to 300, 10 to 400, 10 to 500, 10 to 600, 10 to 700, 10 to 800, 10 to 900, or 10 to all of the amino acids of the protease having a sequence as set forth in any one of SEQ ID NOs: 1-12, 57-58, and 718. The sequence lengths listed include each full-length protease of SEQ ID NOs: 1-12, 57-58, and 718, as well as each of the shorter length proteases listed, even excluding proteases having more than 900 amino acids. For example, a length of 6 amino acids, a length of 10 to 50, 10 to 100, 10 to 150, 10 to 300, 10 to 400, and 10 to all amino acids can be applied to a sequence having 453 amino acids. The length ranges of amino acid sequences recited herein include the length of each amino acid sequence within the range, with the endpoints included. The recited lengths of amino acid sequences can begin at any single position in the reference sequence, where sufficient amino acids follow the single position to provide the recited length, and the range of sequence lengths can be extended by adding 10 to 100N amino acids, where N = 10 or a larger integer.

A portion of the amino acid sequence or protease may be a target protease and may have at least 40% of the activity of the amino acid sequence or protease.

The intein can be any intein. An intein is a polypeptide that has the ability to cleave itself from a protein after translation and can mediate the ligation of remaining protein fragments (exteins) and can have the ability to cleave DNA at specific sites for their proliferation. The intein can be modified. A modified intein can have the ability to cleave itself but may lose the ability to cleave DNA. Inteins can be, but are not limited to, mTth, Pho_RadA, Tko_RadA, Sce_VMA, mVMA, and Pab_Lon. In this article, intein sequences in intein-modified proteases can be found in InBase, an intein database ( http://www.neb.com/neb/inteins.html ; Perler et al., 1992 Proc Natl Acad Sci USA 89:5577), which is incorporated herein by reference as if fully set forth. The intein in the intein-modified protease may include, but is not limited to, the following intein: APMVPol (Acanthomoeba polyphaga Mimivirus), AbrPRP8 (Aspergillus brevipes FRR2439), Aca-JER2004PRP8 (Ajellomyces capsulatus), Aca-H143PRP8 (Histoplasma capsulatus H143), Ade-ER3PRP8 (Ajellomyces dermatitidis ER-3), Aca-NAm1PRP8 (Histoplasma capsulatus NAm1), Afu-Af293PRP8 (Aspergillus fumigatus var. ellipticus strain 14081), Af293), Ade-SLH14081PRP8 (Histoplasma dermatitidis SLH14081), Afu-FRR0163PRP8 (Aspergillus fumigatus strain FRR0163), Afu-NRRL5109PRP8 (Aspergillus fumigatus ellipsoidal variant NRRL 5109), Ani-FGSCA4PRP8 (Aspergillus nidulans FGSC A), Agi-NRRL6136PRP8 (Aspergillus giganteus strain NRRL 6136), AviPRP8 (Aspergillus viridinutans strain FRR0577), BciPRP8 (Botrytis cinerea strain FRR0577), and cinerea), Bde-JEL423PRP8-1 (Batrachochytrium dendrobatidis JEL423), Bde-JEL197RPB2 (Batrachochytrium dendrobatidis JEL197), Bde-JEL423eIF-5B (Batrachochytrium dendrobatidis JEL423), Bde-JEL423PRP8-2 (Batrachochytrium dendrobatidis JEL423), Bfu-B05PRP8 (Botryotinia fuckeliana B05.10), Bde-JEL423RPC2 (Batrachochytrium dendrobatidis JEL423), CIVRIR1 (Chilo iridescent virus), CV-NY2AORF212392 (Chlorella virus NY2A, virusNY2A), CV-NY2ARIR1 (Chlorella virus NY2A), CZIVRIR1 (Costelytra zealandica iridescent virus), Cba-WM02.98PRP8 (Cryptococcus bacillisporus strain WM02.98), Cba-WM728PRP8 (Cryptococcus bacillisporus strain WM728), CeuClpP (Chlamydomonas eugametos), CgaPRP8 (Cryptococcus gattii), ClaPRP8 (Cryptococcus laurentii strain CBS139), CmoClpP (Chlamydomonas moewusii strain UTEX 97) 97), CmoRPB2 (green algae UTEX 97 strain), CglVMA (Candida glabrata), CpaThrRS (Candida parapsilosis strain CLIB214), Fne-APRP8 (Filobasidiella neoformans Serotype A), Cne-JEC21PRP8 (Cryptococcus neoformans JEC21), Fne-ADPRP8 (Cryptococcus neoformans Serotype AD), CreRPB2 (Chlamydomonas reinhardtii), CroVRPB2 (Cafeteria roenbergensis virus BV-PW1) BV-PW1), CroVRIR1 (Lombjor restaurant bug virus BV-PW1), CroVPol (Lombjor restaurant bug virus BV-PW1), CroVTop2 (Lombjor restaurant bug virus BV-PW1), CtrThrRS (Candida tropicalis ATCC750), CstRPB2 (Coelomomyces stegomyiae), CtrVMA (Candida tropicalis), DdiRPC2 (Dictyostelium discoideum strain AX4), DhanVMA (Debaryomyces hansenii CBS767), CBS767), Ctr-MYA3404VMA (Candida tropicalis MYA-3404), DhanGLT1 (Debaryomyces hansenii CBS767), FteRPB2 (Floydiella terrestris strain UTEX 1709), GthDnaB (Guillardia theta), EniPRP8 (Emericella nidulans R20), Eni-FCSGA4PRP8 (Emericella nidulans FGSC A4), HaV01Pol (Heterosigma akashiwo virus 01), HcaPRP8 (Histoplasma capsulatum), IIV6RIR1 (Invertebrate iridescent virus 6) 6), Kex-CBS379VMA (Kazachstania exiguastrain CBS379), Kla-CBS683VMA (Kluyveromyces lactisstrain CBS683), Kla-IFO1267VMA (Kluyveromyces lactis IFO1267), Kla-NRRLY1140VMA (Kluyveromyces lactis NRRL Y-1140), LelVMA (Lodderomyces elongisporus), NauPRP8 (Neosartorya aurata NRRL 4378), Mca-CBS113480PRP8 (Microsporum canis CBS 113480, Microsporum canis CBS 113480), NfiPRP8 (Neosartorya afischeri), Nfe-NRRL5534PRP8 (Neosartorya fennelliae NRRL 5534), Ngl-FRR1833PRP8 (Neosartorya glabra FRR1833), Ngl-FR2163PRP8 (Neosartorya glabra FRR2163), NquPRP8 (Neosartorya quadricincta strain NRRL 4175), NspiPRP8 (Neosartorya spinosa FRR4595, Neosartorya aspinosa) FRR4595), Pabr-Pb01PRP8 (Paracoccidioides brasiliensis Pb01), Pabr-Pb03PRP8 (Paracoccidioides brasiliensis Pb03), PanGLT1 (Podospora anserina), PanCHS2 (Podospora anserina), PchPRP8 (Penicillium chrysogenum), PblPRP8-a (Phycomyces blakesleeanus), Pbr-Pb18PRP8 (Paracoccidioides brasiliensis Pb18), PblPRP8-b (PexPRP8 (Penicillium expansum), PguGLT1 (Pichia guilliermondii), guilliermondii), PnoGLT1 (Phaeosphaeria nodorumSN15), Pgu-altGLT1 (Pichia guilliermondii), PstVMA (Pichia stipitisCBS 6054), PnoRPA2 (Philips guilliermondiiSN15), PpuDnaB (Porphyra purpurea), PtrPRP8 (Pyrenophora tritici-repentis Pt-1C-BF), PvuPRP8 (Penicillium vulpinum), PyeDnaB (Porphyra yezoensis), Sca-CBS4309VMA (Budding Yeast CBS4309 strain, Saccharomyces castellii strain CBS4309), SasRPB2 (Spiromyces aspiralis NRRL 22631), SceVMA, VMA (Saccharomyces cerevisiae), Sca-IFO1992VMA (Saccharomyces castellii strain IFO1992), Sce-DH1-1AVMA (Saccharomyces cerevisiae DH1-1A), ScarVMA (Saccharomyces cariocanus strain UFRJ 50791), 50791), Sce-Jay291VMA (Saccharomyces cerevisiae JAY291), Sce-YJM789VMA (Saccharomyces cerevisiae YJM789 strain), Sce-OUT7091VMA (Saccharomyces cerevisiae OUT7091), Sce-OUT7112VMA (Saccharomyces cerevisiae OUT7112), SjaVMA (Schizosaccharomyces japonicus yFS275), Sex-IFO1128VMA (Saccharomyces exiguus strain IFO1128), SheRPB2 (Stigeoclonium helveticum strain UTEX 441), SdaVMA (Saccharomyces dairenensis strain CBS 421), 421), SpaVMA (Saccharomyces pastorianus IFO11023), SpuPRP8 (Spizellomyces punctatus), SunVMA (Saccharomyces cerevisiae CBS 398 strain, Saccharomyces unisporus strain CBS398), TglVMA (Torulaspora globosa strain CBS 764), TprVMA (Torulaspora pretoriensis strain CBS 5080), Ure-1704PRP8 (non-pathogenic fungus, Uncinocarpus reesii), VpoVMA (Vanderwaltozyma polyspora strain CBS 2163), 2163), WIVRIR1 (Wiseanairidescent virus), ZroVMA (Zygosaccharomyces rouxii strain CBS 688), ZbiVMA (Zygosaccharomyces bisporus strain CBS 702), ZbaVMA (Zygosaccharomyces bailii strain CBS 685), AP-APSE1dpol (Acyrthosiphon pisum secondary endosymbiot phage 1), AP-APSE2dpol (Bacteriophage APSE-2), AP-APSE4dpol (Bacteriophage of Candidatus Hamiltonella 5ATac strain), defensastrain 5ATac bacteriophage), AP-APSE5dpol (bacteriophage APSE-5, Bacteriophage APSE-5), AP-Aaphi23MupF (bacteriophage Aaphi23, Bacteriophage Aaphi23), AaeRIR2 (Aquifex aeolicus strain VF5), Aave-AAC001RIR1 (Acidovoraxavenae subsp. citrulli AAC00-1), Aave-AAC001Aave1721 (Bacterial fruit spot pathogen AAC00-1), Aave-ATCC19860RIR1 (Bacterial fruit spot pathogen ATCC 19860), AbaHyp-02185 (Acinetobacter baumannii ACICU, Acinetobacter baumannii ACICU), AceRIR1 (Acidothermus cellulolyticus 11B), AehDnaB-1 (Alkalilimnicola ehrlichei MLHE-1), AehDnaB-2 (Alkalilimnicola ehrlichei MLHE-1), AehRir1 (Alkalilimnicola ehrlichei MLHE-1), MupFMupF (Aggregatibacter phage S1249), AhaDnaE-c (Aphanothece halophytica), AhaDnaE-n (Halophilic Rhizoctonia solani), Alvi-DSM180GyrA (Allochromatium vinosum DSM180), 180), AmaMADE823 (Alteromonas macleodii), Amax-CS328DnaX (Arthrospira maxima CS-328), AovDnaE-c (Aphanizomenon ovalisporum), AovDnaE-n (Aphanizomenon ovalisporum), Apl-C1DnaX (Arthrospira platensis), AspDnaE-c (Anabaena species PCC7120), Arsp-FB24DnaB (Arthrobacter species FB24), AspDnaE-n (Anabaena PCC7120), AvaDnaE-c (Anabaena variabilis ATCC29413). ATCC29413), AvinRIR1BIL (Azotobacter vinelandii), AvaDnaE-n (Anabaena variabilis ATCC29413), Bce-MCO3DnaB (Burkholderia cenocepacia MC0-3), Bce-PC184DnaB (Burkholderia PC184), Bse-MLS10TerA (Bacillus selenitireducens MLS10), BsuP-M1918RIR1 (B. subtilis M1918 prophage), BsuP-SPBc2RIR1 (B. subtilis strain 168Sp 8 prophage), BsuP-SPBc2RIR1 (B. subtilis strain 168Sp beta 8 prophage). c2prophage), Bcep1808_7358 (Burkholderia vietnamiensis G4), CP-P1201Thy1 (Corynebacterium phage P1201), CagRIR1 (Chlorochromatium aggregatum), CauSpoVR (Chloroflexus aurantiacus J-10-fl), CbP-C-StRNR (Clostridium botulinum phage C-St), CbP-D1873RNR (Botulinum phage D), Cbu-DugwayDnaB (Coxiella burnetii Dugway 5J108-111). 5J108-111), Cbu--GoatDnaB (Coxiella burnetii MSU Goat Q177), Cbu-RSA334DnaB (Coxiella burnetii RSA334), Cbu-RSA493DnaB (Coxiella burnetii RSA 493), CceHyp1-Csp-2 (Cyanothece sp. ATCC 51142), CchRIR1 (Chlorobium chlorochromatii CaD3), CcyHyp1-Csp-1 (Cyanothece sp. CCY0110, Cyanothece sp. sp.CCY0110), CcyHyp1-Csp-2(Cytotrichum cyanobacteria CCY0110), Cfl-DSM20109DnaB(Cellulomonas flavigenaDSM20109), ChyRIR1(Carboxydothermus hydrogenoformans Z-2901), CklPTerm(Clostridium kluyveri DSM 555), Cra-CS505DnaE-c(Cylindrospermopsis raciborskii CS-505), CS-505), Cra-CS505DnaE-n (Cylindrospermum CS-505), Cra-CS505GyrB (Cylindrospermum CS-505), Csp-CCY0110DnaE-c (Cylindrospermum CCY0110), Csp-CCY0110DnaE-n (Cylindrospermum CCY0110), Csp-PCC7424DnaE-c (Cylindrospermum PCC7424), Csp-PCC7424DnaE-n (Cylindrospermum PCC 7424), Csp-PCC7425DnaB (Cylindrospermum PCC 7425), Csp-PCC7822DnaE-n (Cylindrospermum PCC 7822), Csp-PCC8801DnaE-c (Cylindrospermum PCC 8801), Csp-PCC8801DnaE-n (Cyanobacterium PCC 8801), CthATPaseBIL (Clostridium thermocellum), Cth-ATCC27405TerA (Clostridium thermocellum ATCC27405), Cth-DSM2360TerA (Clostridium thermocellum DSM 2360), CwaDnaB (Crocosphaera watsonii WH 8501), CwaDnaE-c (Crocosphaera watsonii WH 8501), CwaDnaE-n (Crocosphaera watsonii WH 8501), CwaPEP (Crocosphaera watsonii WH8501), CwaRIR1 (Crocosphaera watsonii WH 8501). 8501), DaudRIR1 (Candidatus Desulforudis audaxviator MP104C), DgeDnaB (Deinococcus geothermalis DSM11300), Dha-DCB2RIR1 (Desulfitobacterium hafniense DCB-2), Dha-Y51RIR1 (Halodesulfitobacterium Y51), Dpr-MLMS1RIR1 (delta proteobacterium MLMS-1), DraRIR1 (Deinococcus radiodurans R1TIGR strain), strain), DraSnf2-c (Deinococcus radiodurans R1 TIGR strain), Snf2-nN-TERM (Deinococcus radiodurans R1 TIGR strain), Dra-ATCC13939Snf2 (Deinococcus radiodurans R1 ATCC13939 Brooks & Murray strain), UDPGD (Dictyoglomus thermophilum H-6-12), DvulParB (Desulfovibrio vulgaris subsp. vulgaris DP4), EP-Min27Primase (Enterobacteria phage Min27), FalDnaB (Frankia alni ACN14a), ACN14a), Fsp-CcI3RIR1 (Frankia species CcI3), GobDnaE (Gemmata obscuriglobus UQM2246), GobHyp (Gemmata obscuriglobus UQM2246), GviDnaB (Gloeobacter violaceus PCC 7421), GviRIR1-2 (Gloeobacter violaceus PCC 7421), GviRIR1-1 (Gloeobacter violaceus PCC 7421), HhalDnaB (Halorhodospira halophila SL1, Halophila halophila SL1), Kfl-DSM17836DnaB (Kribbella flavida DSM 17836), KraDnaB (Kineococcus radiotolerans SRS30216), LLP-KSY1PolA (Lactococcus phage KSY1), LP-phiHSIChelicase (Listonella pelagia phage phiHSIC), Lsp-PCC8106GyrB (Lyngbya sp. PCC 8106), MP-BeDnaB (Mycobacterium bethlehem phage Bethlehem), MP-Begp51 (Mycobacteriophage Bethlehem), MP-Cateragp206 (Mycobacteriophage Catera), MP-KBGgp53 (Mycobacterium phage KBG), MP-OmegaDnaB (Mycobacteriophage Omega), MP-Mcjw1DnaB (Mycobacteriophage CJW1), gp50 (Mycobacteriophage U2), Maer-NIES843DnaB (Microcystis aeruginosa NIES-843). NIES-843), Maer-NIES843DnaE-c (Microcystis aeruginosa NIES-843), Maer-NIES843DnaE-n (Microcystis aeruginosa NIES-843), Mau-ATCC27029GyrA (Micromonospora aurantiaca ATCC 27029), Mav-104DnaB (Mycobacterium avium 104), Mav-ATCC25291DnaB (Mycobacterium avium subsp.avium ATCC 25291), Mav-ATCC25292DnaB (Mycobacterium avium subsp.avium ATCC 25291), Mav-ATCC25293DnaB (Mycobacterium avium subsp.avium ATCC 25293), Mav-ATCC25294DnaB (Mycobacterium avium subsp.avium ATCC 25293), Mav-ATCC25295DnaB (Mycobacterium avium subsp.avium ATCC 25293), Mav-ATCC25296DnaB (Mycobacterium avium subsp.avium ATCC 25291), Mav-ATCC35712DnaB (Mycobacterium avium subsp. paratuberculosis str.k10), MboPps1 (Mycobacterium bovis subsp. bovis AF2122/97), MboRecA (Mycobacterium bovis subsp. AF2122/97), MboPps1 (Mycobacterium bovis subsp. bovis AF2122/97), Mbo-AF2122DnaB (Mycobacterium bovis subsp. AF2122/97), Mbo-1173PDnaB (Mycobacterium bovis BCG Pasteur 1173P, Mycobacterium bovis BCG Pasteur 1173P), McaMupF (Methylococcus capsulatus Bath prophage MuMc02), McaRIR1 (Methylococcus capsulatus Bath), MchRecA (Mycobacterium chitae), Mcht-PCC7420DnaE-1 (Microcoleuschthonoplastes PCC7420), Mcht-PCC7420DnaE-2c (Microcoleuschthonoplastes PCC7420). PCC7420), Mcht-PCC7420DnaE-2n(Microcoleus spp. PCC7420), Mcht-PCC7420GyrB(Microcoleus spp. PCC7420), Mcht-PCC7420RIR1-1(Microcoleus spp. PCC7420), Mcht-PCC7420RIR1-2(Microcoleus spp. PCC7420), Mexhelicase(Methylobacterium extorquens AM1), MexTrbC(Methylobacterium extorquens AM1), MfaRecA(Mycobacterium fallax), MflGyrA(Mycobacterium flavescens Fla0), flavescensFla0), MflRecA (Mycobacterium flavescens Fla0), Mfl-ATCC14474RecA (Mycobacterium flavescens ATCC14474), Mfl-PYR-GCKDnaB (Mycobacterium flavescens PYR-GCK), MgaGyrA (Mycobacterium gastri), MgaRecA (Mycobacterium gastri), MgaPps1 (Mycobacterium gastri), Mgi-PYR-GCKDnaB (Mycobacterium gilvum PYR-GCK), Mgi-PYR-GCKGyrA (Mycobacterium flavescens PYR-GCK), MgoGyrA (Mycobacterium gordonii), gordonae), Min-1442DnaB (Mycobacterium intracellulare), Min-ATCC13950GyrA (Mycobacterium intracellulare ATCC 13950), MkasGyrA (Mycobacterium kansasii), Mkas-ATCC12478GyrA (Mycobacterium kansasii ATCC 12478), Mle-Br4923GyrA (Mycobacterium leprae Br4923), leprae TN strain), Mle-TNRecA (Mycobacterium leprae TN strain), MmaGyrA (Mycobacterium malmoense), MmagMagn8951BIL (Magnetospirillum magnetotacticum MS-1), MshRecA (Mycobacterium shimodei), MsmDnaB-1 (Mycobacterium smegmatis MC2 155), MsmDnaB-2 (Mycobacterium smegmatis MC2 155), Msp-KMSDnaB (Mycobacterium species KMS), Msp-KMSDnaB (Mycobacterium species Mtu-CRecA (Mycobacterium tuberculosis C), Mtu-CPHLRecA (Mycobacterium tuberculosis CPHL_A), Mtu-EAS054RecA (Mycobacterium tuberculosis EAS054), Mtu-CanettiRecA (Mycobacterium tuberculosis Canetti strain, Mycobacterium tuberculosis strain Canetti), Mtu-F11DnaB (Mycobacterium tuberculosis F11 strain), Mtu-H37RaDnaB (Mycobacterium tuberculosis H37Ra), Mtu-H37RvDnaB (Mycobacterium tuberculosis H37Rv), Mtu-H37RvRecA (Mycobacterium tuberculosis H37Rv and CDC1551), Mtu-HaarlemDnaB (Mycobacterium tuberculosis Haarlem strain, Mycobacterium tuberculosisstr.Haarlem), Mtu-R604RecA-n (Mycobacterium tuberculosis 98-R604INH-RIF-EM), Mtu-K85RecA (Mycobacterium tuberculosis K85), Mtu-So93RecA (Mycobacterium tuberculosis So93/canetti subsp.), Mtu-T17RecA-c (Mycobacterium tuberculosis T17), Mtu-T17RecA-n (Mycobacterium tuberculosis T17), Mtu-T46RecA (Mycobacterium tuberculosis T46), Mtu-T85RecA (Mycobacterium tuberculosis T85), MvanDnaB (Mycobacterium vanbaalenii PYR-1, Mycobacterium vanbaalenii PYR-1), Mtu-T92RecA (Mycobacterium tuberculosis T92), MvanGyrA (Mycobacterium van Barenyi PYR-1), MxaRAD25 (Myxococcus xanthus DK1622), MxeGyrA (Mycobacterium xenopi strain IMM5024), Naz-0708RIR1-2 (Nostoc azollae 0708), Naz-0708RIR1-1 (Nostoc azollae 0708), NfaDnaB (Nocardia farcinica IFM 10152), 10152), NfaNfa15250 (Nocardia spp. IFM10152), NfaRIR1 (Nocardia spp. IFM 10152), Nosp-CCY9414DnaE-n (Nodularia spumigena CCY9414), NpuDnaB (Nostoc punctiforme), NpuGyrB (Nostoc punctiforme), Npu-PCC73102DnaE-c (Nostoc punctiforme PCC73102), Npu-PCC73102DnaE-n (Nostoc punctiforme PCC73102), Nsp-JS614DnaB (Nocardioides species JS614) JS614), Nsp-JS614TOPRIM (Nocardia species JS614), Nsp-PCC7120DnaB (Nostoc species PCC7120), Nsp-PCC7120DnaE-c (Nostoc species PCC7120), Nsp-PCC7120DnaE-n (Nostoc species PCC7120), Nsp-PCC7120RIR1 (Nostoc species PCC7120), OliDnaE-c (Oscillatorialimnetica str. Solar Lake), OliDnaE-n (Oscillatorialimnetica solar lake), PP-PhiELHelicase (Pseudomonas aeruginosa phage PhiEL), PP-PhiELORF11 (Pseudomonas aeruginosa phage), PP-PhiELORF40 (Pseudomonas aeruginosa phage), PP-PhiELORF39 (Pseudomonas aeruginosa phage), PflFhaBIL (Pseudomonas fluorescens Pf-5), Pma-ExH1DnaE (Persephonella marina EX-H1), PlutRIR1 (Pelodictyon luteolum DSM 273), Pma-EXH1GyrA (Persephonella marina EX-H1), PnaRIR1 (Polaromonas naphthalenivorans CJ2), CJ2), Posp-JS666DnaB (Polarmonas species JS666), PuncDnaB (Polynucleobacter sp. QLW-P1DMWA-1), Posp-JS666RIR1 (Polarmonas species JS666), Pssp-A1-1Fha (Pseudomonas species A1-1), PsyFha (Pseudomonas syringae pv. tomatostr. DC3000), Rbr-D9GyrB (Raphidiopsis brookii D9), RceRIR1 (Rhodospirillum centenum SW SW), Rer-SK121DnaB (Rhodococcus erythropolis SK121), RmaDnaB (Rhodothermus marinus), Rma-DSM4252DnaE (Rhodothermus marinus DSM 4252), Rma-DSM4252DnaB (Rhodothermus marinus DSM4252), RspRir1 (Roseovarius species 217), SaP-SETP12dpol (Salmonella phage SETP12), SETP12), SaP-SETP3Helicase (Salmonella phage SETP3), SaP-SETP3dpol (Salmonella phage SETP3), SaP-SETP5dpol (Salmonella phage SETP5), SareDnaB (Salinispora arenicola CNS-205), ReGHelicase (Streptomyces avermitilis MA-4680), Sel-PC6301RIR1 (Synechococcus elongatus PCC6301), 6301), Sel-PC7942DnaE-c (Synechococcus elongatus PC7942), Sel-PC7942RIR1 (Synechococcus elongatus PC7942), Sel-PC7942DnaE-n (Synechococcus elongatus PC7942), Sel-PCC6301DnaE-n (Synechococcus elongatus PCC 6301), Sel-PCC6301DnaE-c (Synechococcus elongatus PCC 6301 and PCC7942), ShP-Sfv-2a-2457T-nPrimase (2a Shigella flexneri 2astr.2457T), SepRIR1 (Staphylococcus epidermidis RP62A, Staphylococcus epidermidis RP62A), ShP-Sfv-2a-301Primase (2a Shigella flexneri 301 strain), ShP-Sfv-5Primase (5 Shigella flexneri 8401 strain, Shigella flexneri 5str.8401), SoP-SO1dpol (Sodalis phage SO-1), SruDnaB (Salinibacter ruber DSM 13855), SplDnaX (Spirulina platensis strain C1), SruPolBc (Salinibacter ruber DSM 13855), SruRIR1 (Salinibacterium ruber DSM 13855), SspDnaB (Synechocystis species strain PCC6803), PCC6803), SspDnaE-n, DnaE-N (Synechococcus species PCC6803), SspDnaE-c, DnaE-C (Synechococcus species PCC6803), SspDnaX (Synechococcus species PCC6803), Ssp-JA2RIR1 (Synechococcus species JA-2-3B a 2-13, Synechococcus species JA-2-3B a 2-13), Ssp-JA2DnaB (Synechococcus species JA-2-3B a 2-13), SspGyrB (Synechococcus PCC6803), Ssp-JA3DnaB (Synechococcus species JA-3-3Ab), Ssp-JA3RIR1 (Synechococcus species JA-3-3Ab), Ssp-PCC7002DnaE-c (Synechococcus PCC 7002 strain), Ssp-PCC7002DnaE-n (Synechocystis PCC 7002 strain), Ssp-PCC7335RIR1 (Synechococcus PCC 7335), StP-TwortORF6 (Staphylococcus phage Twort), Susp-NBC371DnaB (Sulfurovum sp. NBC37-1), Taq-Y51MC23DnaE (Thermus aquaticus Y51MC23), TelDnaE-c (Thermo synechoccuse longatus BP-1), Tcu-DSM43183RecA (Thermomonospora curvata DSM 43183), 43183), TelDnaE-n (Thermosynechococcus elongatus BP-1), Taq-Y51MC23RIR1 (Thermus aquaticus Y51MC23), TerDnaB-1 (Trichodesmium erythraeum IMS101), IMS101), TerDnaB-2 (Trichoderma rubripes IMS101), TerDnaE-2 (Trichoderma rubripes IMS101), TerDnaE-1 (Trichoderma rubripes IMS101), TerDnaE-3c (Trichoderma rubripes IMS101), TerDnaE-3n (Trichoderma rubripes IMS101), TerGyrB (Trichoderma rubripes IMS101), TerNdse-1 (Trichoderma rubripes IMS101), TerNdse -2 (Trichoderma rubripes IMS101), TerRIR-1 (Trichoderma rubripes IMS101), TerRIR-2 (Trichoderma rubripes IMS101), TerRIR-3 (Trichoderma rubripes IMS101), TerRIR-4 (Trichoderma rubripes IMS101), TerSnf2 (Trichoderma rubripes IMS101), TerThyX (Trichoderma rubripes IMS101), TfusRecA-1 (Thermobifida YX, Thermobifida fusca YX), TfusRecA-2 (brown thermophilic bacteria YX), TfusTfu2914 (brown thermophilic bacteria YX), Thsp-K90RIR1 (halophilic alkaliphilic sulfur oxidizing bacteria K90mix, Thioalkalivibrio sp.K90mix), Tth-DSM571RIR1 (thermoanaerobacteriumthermosaccharolyticum DSM 571), Tth-HB27DnaE-1, Tth (Thermusthermophilus HB27, Thermusthermophilus HB27), Tth-HB27DnaE-2 (Thermus thermophilus HB27), Tth-HB27RIR1-1 (Thermus thermophilus HB27), Tth-HB27RIR1-2 (Thermus thermophilus HB27), Tth-HB8DnaE-1 (Thermus thermophilus HB8), Tth-HB8DnaE-2 (Thermus thermophilus HB8), Tth-HB8RIR1-1 (Thermus thermophilus HB8), Tth-HB8RIR1-2 (Thermus thermophilus HB8), TvuDnaE-c (Thermosynechococcus vulcanus), TvuDnaE-n (Thermosynechococcus vulcanus), TyeRNR-1 (Thermodesulfovibrio yellowstonii DSM11347, Thermodesulfovibrio yellowstonii DSM11347), TyeRNR-2 (Thermosulfuric Acidovibrio huangshiensis DSM11347), ApeAPE0745 (Aeropyrumpernix K1), Cme-booPol-II (Candidatus Methanoregulaboonei 6A8), Fac-Fer1RIR1 (Ferroplasma acidarmanus taxon: 97393), FacPps1 (Ferroplasma acidarmanus), Fac-TypeIRIR1 (Ferroplasma acidarmanus type I), FacPps1 (Ferroplasma acidarmanus), HmaCDC21 (Haloarcula marismortui ATCC 43049, Haloarcula marismortui ATCC 43049), HmaPol-II (Halobacterium maritima ATCC 43049), HmaPolB (Halobacterium maritima ATCC 43049), HmaTopA (Halobacterium maritima ATCC 43049), Hmu-DSM12286MCM (Halomicrobium mukohataei DSM 12286), Hmu-DSM12286PolB (Halomicrobium mukohataei DSM 12286), Hsa-R1MCM (Halobacterium salinarum R-1), Hsp-NRC1CDC21 (Halobacterium species NRC-1), Hsp-NRC1Pol-II (Halobacterium salinarum NRC-1 NRC-1), HutMCM-2 (Halorhabdusutahensis DSM 12940), HutMCM-1 (Halorhabdusutahensis DSM 12940), HwaGyrB (Haloquadratum walsbyi DSM 16790), HvoPolB (Haloferax volcanii DS70), HwaMCM-1 (Haloferax volcanii DSM 16790), HwaMCM-2 (Haloferax volcanii DSM 16790), HwaMCM-3 (Haloferax volcanii DSM 16790), HwaMCM-4 (Haloferax volcanii DSM 16790), HwaPol-II-1 (Halophilic archaea vosbi DSM 16790), HwaPol-II-2 (Halophilic archaea vosbi DSM 16790), HwaPolB-1 (Halophilic archaea vosbi DSM 16790), HwaPolB-2 (Halophilic archaea vosbi DSM 16790), HwaPolB-3 (Halophilic archaea vosbi DSM 16790), HwaRCF (Halophilic archaea vosbi DSM 16790), HwaRIR1-1 (Halophilic archaea vosbi DSM 16790), HwaRIR1-2 (Halophilic archaea vosbi DSM 16790), HwaTop6B (Halophilic archaea vosbi DSM 16790), rPolA (Halophilic archaea vosbi DSM 16790), MaeoPol-II (Methanococcus aeolicus Nankai-3), MaeoRFC (Methanococcus Nankai-3), MaeoRNR (Methanococcus Nankai-3), Maeo-N3Helicase (Methanococcus Nankai-3), UDPGD (Methanococcus Nankai-3), Maeo-N3RtcB (Methanococcus Nankai-3), Mein-MEPEP (Methanocaldococcus infernus ME), Mein-MERFC (Methanococcus infernus ME), MemarMCM2 (Methanocaldococcus marisnigri JR1 JR1), MemarPol-II (Methanococcus fervensis JR1), Mesp-FS406PolB-1 (Methanocaldococcus sp. FS406-22), Mesp-FS406PolB-2 (Methanococcus sp. FS406-22), Mesp-FS406PolB-3 (Methanococcus sp. FS406-22), Msp-FS406-22LHR (Methanococcus sp. FS406-22), Mfe-AG86Pol-1 (Luminescent Methanococcus sp. AG86, Methanocaldococcus fervens AG86), Mfe-AG86Pol-2 (Luminescent Methanococcus sp. AG86), MhuPol-II (Methanospirillum hungateii JF-1, Methanospirillum hungateii JF-1), MjaGF-6P (Methanococcus jannaschii), MjaHelicase (Methanococcus jannaschii), MjaHyp-1 (Methanococcus jannaschii), MjaIF2 (Methanococcus jannaschii), MjaKlba (Methanococcus jannaschii), MjaPEP (Methanococcus jannaschii), MjaPol-1 (Methanococcus jannaschii), MjaPol-2 (Methanococcus jannaschii), Mj aRFC-1 (Methanococcus jannaschii), MjaRFC-2 (Methanococcus jannaschii), MjaRFC-3 (Methanococcus jannaschii), MjaRNR-1 (Methanococcus jannaschii), MjaRNR-2 (Methanococcus jannaschii), MjaHyp-2 (Methanococcus jannaschii), MjaTFIIB (Methanococcus jannaschii), UDPGD (Methanococcus jannaschii), Mjar-Gyr (Methanococcus jannaschii), rPolA' (Methanococcus jannaschii), Mja rPol A' (Methanococcus jannaschii), MkaCDC48 (Methanopyrus kandleri AV19), MkaEF2 (Methanothermus kandleri AV19), MkaRFC (Methanothermus kandleri AV19), MkaRtcB (Methanothermus kandleri AV19), MkaVatB (Methanothermus kandleri AV19), MthRIR1 (Methanothermobacter thermautotrophicus), Mvu-M7Helicase (Methanocaldococcus vulcanius M7), M7), Mvu-M7Pol-1 (Methylated Sophorae volcanica M7), Mvu-M7Pol-2 (Methylated Sophorae volcanica M7), Mvu-M7Pol-3 (Methylated Sophorae volcanica M7), UDPGD (Methylated Sophorae volcanica M7), NeqPol-c (Nanoarchaeum equitans Kin4-M), NeqPol-n (Nanoarchaeum equitans Kin4-M), Nma-ATCC43099MCM (Natrialba magadii ATCC 43099), Nma-ATCC43099PolB-1 (Natrialba magadii ATCC 43099), Nma-ATCC43099PolB-2 (Natrialba magadii ATCC 43099), NphCDC21 (Natronomonas pharaonis DSM 2160, Natronomonas pharaonis DSM2160), NphPolB-2 (Natronomonas pharaonis DSM 2160), NphPolB-1 (Natronomonas pharaonis DSM 2160), rPolA" (Natronomonas pharaonis DSM 2160), PabCDC21-1 (Pyrococcus abyssi), Pa bCDC21-2 (Thermococcus aphrodisiacs), PabIF2 (Thermococcus aphrodisiacs), PabKlbA (Thermococcus aphrodisiacs), PabLon (Thermococcus aphrodisiacs), PabMoaa (Thermococcus aphrodisiacs), PabPol-II (Thermococcus aphrodisiacs), PabRFC-1 (Thermococcus aphrodisiacs), PabRFC-2 (Thermococcus aphrodisiacs), PabRIR1-1 (Thermococcus aphrodisiacs), PabRIR1-2 (Thermococcus aphrodisiacs), PabRIR1-3 (Thermococcus aphrodisiacs), PabHyp-2 (Thermococcus aphrodisiacs), PabVMA (Thermococcus aphrodisiacs), ParRIR1 (Pyrobaculum arsenaticum DSM 13514), PfuCDC21 (Pyrococcus furiosus), furiosus), PfuIF2 (Pyrococcus furiosus), PfuKlbA (Pyrococcus furiosus), PfuLon (Pyrococcus furiosus), PfuRFC (Pyrococcus furiosus), PfuRIR1-1 (Pyrococcus furiosus), PfuRIR1-2 (Pyrococcus furiosus), PfuHyp-2 (Pyrococcus furiosus), PfuTopA (Pyrococcus furiosus), PfuVMA (Pyrococcus furiosus), PhoCDC21-1 (Pyrococcus horikoshii OT3, Pyrococcus horikoshii OT3), PhoCDC21-2 (Thermococcus holmenii OT3), PhoIF2 (Thermococcus holmenii OT3), PhoKlbA (Thermococcus holmenii OT3), PhoLHR (Thermococcus holmenii OT3), PhoLon (Thermococcus holmenii OT3), PolI (Thermococcus holmenii OT3), PhoPol-II (Thermococcus holmenii OT3), PhoRFC (Thermococcus holmenii OT3), PhoRIR1 (Thermococcus holmenii OT3), PhoRadA (Thermococcus holmenii OT3), PhoVMA (Thermococcus holmenii OT3), PhoHyp-2 (Thermococcus holmenii OT3), Phor-Gyr (Thermococcus holmenii OT3), Psp-GBDPol (Pyrococcus species GB-D, Pyrococcus species GB-D), Smar1471 (Staphylothermus maritima F1, Staphylothermus marinus F1), PtoVMA (Picrophilus torridus DSM 9790), Tac-ATCC25905VMA (Thermoplasma acidophilum ATCC 25905), SmarMCM2 (Staphylococcus marinus F1), Tac-DSM1728VMA (Thermoplasma acidophilum DSM1728), Tsp-TYPol-1 (Thermococcus aggregans), Tsp-TYPol-2 (Thermococcus aggregans), Tsp-TYPol-3 (Thermococcus aggregans), TbaPol-II (Thermococcus barophilus MP), TfuPol-1 (Thermococcus fusneri ...fuPol-2 (Thermococcus fusneri), TfuPol-3 (Thermococcus fusneri), Tsp-TYPol-1 (Thermococcus aggregans), Tsp-TYPol-2 (Thermococcus aggregans), Tsp-TYPol-3 (Thermococcus aggregans), TbaPol-II (Thermococcus barophilus MP), TfuPol-1 (Thermococcus fusneri), TfuPol-2 (Thermococcus fusneri), TfuPol-2 (Thermococcus fusneri), TfuPol-3 (Thermococcus fusneri), TfuPol-1 (Thermococcus fumicolans), ThyPol-1 (Thermococcus hydrothermalis), TfuPol-2 (Thermococcus flexneri), ThyPol-2 (Thermococcus hydrothermalis), TkoCDC21-1 (Thermococcus Kodakaraensis KOD1, Thermococcus kodakaraensis KOD1), TkoCDC21-2 (Thermococcus Koda KOD1), TkoHelicase (Thermococcus Koda KOD1), TkoIF2 (Thermococcus Koda KOD1), TkoKlbA (Thermococcus Koda KOD1) Thermococcus Koda KOD1), TkoLHR (Thermococcus Koda KOD1), Psp-KODPol-1 (Thermococcus Koda KOD1), KODPol-2 (Thermococcus Koda KOD1), TkoPol-II (Thermococcus Koda KOD1), TkoRIR1-1 (Thermococcus Koda KOD1), TkoRFC (Thermococcus Koda KOD1), TkoRIR1-2 (Thermococcus Koda KOD1), TkoRad A (Thermococcus Koda KOD1), TkoTopA (Thermococcus Koda KOD1), Tkor-Gyr (Thermococcus Koda KOD1), TliPol-1 (Thermococcus Koda, Thermococcus litoralis), TliPol-2 (Thermococcus litoralis), TmaPol (Thermococcus marinus), Ton-NA1LHR (Thermococcus onnurineus NA1), Ton-NA1Pol (Thermococcus onnurineus NA1), TpePol (Thermococcus peptonophilus strain SM2), Tsi-MM739Lon (Thermococcus sibiricus MM 739), Tsi-MM739Pol-1 (Thermococcus sibiricus MM 739), Tsi-MM739Pol-2 (Thermococcus sibiricus MM 739), Tsi-MM739RFC (Thermococcus sibiricus MM 739), AM4RtcB (Thermococcus sp. AM4), Tsp-AM4LHR (Thermococcus AM4), Tsp-AM4Lon (Thermococcus AM4), Tsp-AM4RIR1 (Thermococcus AM4), Tsp-GE8Pol-2 (Thermococcus species GE8), Tsp-GE8Pol-1 (Thermococcus GE8), Tsp-GTPol-1 (Thermococcus species GT), Tsp-GTPol-2 (Thermococcus GT), Tsp-OGL-P20Pol (Thermococcus OGL-20P), TthiPol (Thermococcus thioreducens), TziPol (Thermococcus zilligii), TvoVMA (Thermoplasma volcanica GSS1), volcaniumGSS1), Unc-ERSPFL (uncultured archaeon GZfos13E1), Unc-ERSRIR1 (uncultured archaeon GZfos9C4), Unc-MetRFSMCM2 (uncultured archaeon Rice Cluster I), and Unc-ERSRNR (uncultured archaeon GZfos10C7).

The name of the intein provides information about the organism and the name of the protein, giving a homolog of the intein-bearing protein in a well-studied organism. For example, in the name Ade-ER3PRP8, "Ade-ER3" refers to the organism Histoplasma dermatitidis ER-3, and PRP8 is the name of the protein that gives a homolog of the intein-bearing protein in a well-studied organism.

The inteins can be developed to splice under conditional control and can be used as protease switches in non-native protease hosts to modulate protease activity. Libraries of intein-modified proteases can be constructed, expressed in compatible expression hosts, and screened for activity after intein splicing has occurred. This system allows for broad control over screening conditions and, through repeated iterations of mutation and screening, the desired protease properties can be evolved. The intein can be inserted into the protease before (at the amino-terminal end) a serine, threonine, or cysteine. These amino acids can play a role in promoting intein splicing and can be common targets for manipulating inteins into host target proteases that do not otherwise contain an intein sequence. Inteins can be inserted into the protease at any position where a serine, threonine, or cysteine is present in the enzyme's original (or native) amino acid sequence. By inserting a serine, threonine, or cysteine into the sequence, or by mutating the native protease sequence to change the native amino acid to one of these amino acids at any position in the protease, it is possible to place an intein at any desired position within the protease sequence.

In one embodiment, the intein is capable of achieving trans-splicing of an intein-modified protease. The intein can include an N-intein and a C-intein. The amino acid sequence of the N-intein can be at least 70%, 72%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference sequence selected from SEQ ID NO: 38 (DnaE-N), SEQ ID NO: 537 (gp41-1-N), SEQ ID NO: 539 (gp41-8-N), SEQ ID NO: 541 (IMPDH-1-N), and SEQ ID NO: 543 (NrdJ-1-N). The amino acid sequence of the C-intein may be at least 90% identical to a reference sequence selected from the group consisting of SEQ ID NO: 39 (DnaE-C), SEQ ID NO: 538 (gp41-1-C), SEQ ID NO: 540 (gp41-8-C), SEQ ID NO: 542 (IMPDH-1-C), and SEQ ID NO: 544 (NrdJ-1-C).

The intein-modified protease may include a first portion having an N-extein of a target protease and an N-intein of an intein. The carboxyl terminus of the N-extein may be fused to the amino terminus of the N-intein. The first portion may contain a residue selected from the group consisting of SEQ ID NO: 13 (Q53521-T108: DnaE-N), SEQ ID NO: 15 (S154: DnaE-N), SEQ ID NO: 17 (Q53521-S234: DnaE-N), SEQ ID NO: 19 (Q53521-S260: DnaE-N), SEQ ID NO: 21 (Q5321-S263-DnaE-N), SEQ ID NO: 23 (Q53521-T317: DnaE-N), SEQ ID NO: 454 (NI-GG-6H), SEQ ID NO: 456 (S135_IMPDH-NI), SEQ ID NO: 457 (S269_IMPDH-NI), SEQ ID NO: 458 (S293_IMPDH-NI), SEQ ID NO: 460 (S294_IMPDH-NI), SEQ ID NO: 461 (S295_IMPDH-NI), SEQ ID NO: 462 (S296_IMPDH-NI), SEQ ID NO: 463 (S297_IMPDH-NI), SEQ ID NO: 464 (S298_IMPDH-NI), SEQ ID NO: 465 (S299_IMPDH-NI), SEQ ID NO: 466 (S297_IMPDH-NI), SEQ ID NO: 467 (S299_IMPDH-NI), SEQ ID NO: 468 (S299_IMPDH-NI), SEQ ID NO: 469 (S290_IMPDH-NI), SEQ ID NO: 470 (S291_IMPDH-NI), SEQ ID NO: 471 (S293_IMPDH-NI), SEQ ID NO: 472 (S NO: 459 (S317_IMPDH-NI), SEQ ID NO: 460 (T318_IMPDH-NI), SEQ ID NO: 461 (S135_gp41-1-NI), SEQ ID NO: 462 (S269_gp41-1-NI), SEQ ID NO: 463 (S293_gp41-1-NI), SEQ ID NO: 464 (S317_gp41-1-NI), SEQ ID NO: 465 (T318_gp41-1-NI), SEQ ID NO: 466 (S135_gp41-8-NI), SEQ ID NO: 467 (S269_gp41-8-NI), SEQ ID NO: 468 (S293_gp41-8-NI), SEQ ID NO: 469 (S317_gp41-8-NI), SEQ ID NO: 470 (T318_gp41-8-NI), SEQ ID NO: 471 (S135_NrdJ-1-NI), SEQ ID NO: 472 (S269_NrdJ-1-NI), SEQ ID NO: 473 (S293_NrdJ-1-NI), SEQ ID NO: 474 (S317_NrdJ-1-NI), and SEQ ID NO: 475 (T318_NrdJ-1-NI) have sequences that are at least 70%, 72%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the reference sequence.

In one embodiment, the intein-modified protease may include a second portion having a C-intein of the intein and a C-extein of the target protease. The carboxyl terminus of the C-intein may be fused to the amino terminus of the C-extein. The second portion may contain a residue selected from the group consisting of SEQ ID NO: 14 (DnaE-C: T108-Q53521-C), SEQ ID NO: 16 (DnaE-C: S154-Q53521-C), SEQ ID NO: 18 (DnaE-C: S234-Q53521-C), SEQ ID NO: 20 (DnaE-C: S260-Q53521-C), SEQ ID NO: 22 (DnaE-C: S263-Q53521-C), SEQ ID NO: 24 (DnaE-C: T317-Q53521-C), SEQ ID NO: 455 (IC-SUMO-6H), SEQ ID NO: 476 (S135_IMPDH-IC), SEQ ID NO: 477 (S269_IMPDH-IC), SEQ ID NO: 478 (S293_IMPDH-IC), SEQ ID NO: 480 (S394_IMPDH-IC), SEQ ID NO: 481 (S406_IMPDH-IC), SEQ ID NO: 482 (S407_IMPDH-IC), SEQ ID NO: 483 (S408_IMPDH-IC), SEQ ID NO: 484 (S409_IMPDH-IC), SEQ ID NO: 485 (S409_IMPDH-IC), SEQ ID NO: 486 (S408_IMPDH-IC), SEQ ID NO: 487 (S409_IMPDH-IC), SEQ ID NO: 488 (S409_IMPDH-IC), SEQ ID NO: 489 (S410_IMPDH-IC), SEQ ID NO: 490 (S411_IMPDH-IC), SEQ ID NO: 501 (S4 NO: 479 (S317_IMPDH-IC), SEQ ID NO: 480 (T318_IMPDH-IC), SEQ ID NO: 481 (S135_gp41-1-IC), SEQ ID NO: 482 (S269_gp41-1-IC), SEQ ID NO: 483 (S293_gp41-1-IC), SEQ ID NO: 484 (S317_gp41-1-IC), SEQ ID NO: 485 (T318_gp41-1-IC), SEQ ID NO: 486 (S135_gp41-8-IC), SEQ ID NO: 487 (S269_gp41-8-IC), SEQ ID NO: 488 (S293_gp41-8-IC), SEQ ID NO: 489 (S317_gp41-8-IC), SEQ ID The reference sequences in the group consisting of SEQ ID NO: 490 (T318_gp41-8-IC), SEQ ID NO: 491 (S135_NrdJ-1-IC), SEQ ID NO: 492 (S269_NrdJ-1-IC), SEQ ID NO: 493 (S293_NrdJ-1-IC), SEQ ID NO: 494 (S317_NrdJ-1-IC) and SEQ ID NO: 495 (T318_NrdJ-1-IC) have sequences that are at least 70%, 72%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical.

The first and second parts of the intein-modified protease can be separated before splicing. Separation can be achieved by expressing the first and second parts in different compartments of the host cell. Separation can also be achieved by expressing the first and second parts in different host cells. Separation can be achieved by using male and female lines of the same host. The first part can be expressed in a male line of a plant species. The second part can be expressed in a female line of the same plant species. The male and female lines can be crossed during plant breeding to produce lines that have both the first and second parts of the intein-modified protease. Contact of the first part with the second part can cause trans-splicing of the intein-modified protease.

In one embodiment, the intein can achieve cis-splicing of the intein-modified protease. The cis-spliced intein can be internally fused to the target protease. The amino acid sequence of the cis-spliced intein can be selected from the group consisting of SEQ ID NO: 37 (VMA), SEQ ID NO: 40 (Tth), SEQ ID NO: 119 (mTth: EU59 intein), SEQ ID NO: 497 (Cth_ATPase_BIL), SEQ ID NO: 498 (Cwa_RIR1), SEQ ID NO: 499 (Dhan_GLT1), SEQ ID NO: 500 (FSP-CcI3_RIR1), SEQ ID NO: 501 (Gob_Hyp), SEQ ID NO: 502 (Gvi_RIR1-1), SEQ ID NO: 503 (Hhal_DnaB-1), SEQ ID NO: 504 (Hma_CDC21), SEQ ID NO: 505 (Hwa_MCM-1), SEQ ID NO: 506 (Hwa_PolB-2), SEQ ID NO: 507 (Hwa_RIR1-1), SEQ ID NO: NO: 508 (Hwa_RIR1-2), SEQ ID NO: 509 (Hwa_rPol_App), SEQ ID NO: 510 (Kra_DnaB), SEQ ID NO: 511 (Mca_RIR1), SEQ ID NO: 512 (Memar_Pol-II), SEQ ID NO: 513 (Mex_Helicase), SEQ ID NO: 514 (Mhu_Pol-II), SEQ ID NO: 515 (Mja_Klba), SEQ ID NO: 516 (Mja_PEP), SEQ ID NO: 517 (Mja_Pol-2), SEQ ID NO: 518 (Mja_RFC-3), SEQ ID NO: 519 (of Mja_r-Gyr), SEQ ID NO: 520 (MP-Be_gp51), SEQ ID NO: 521 (NSP-PCC7120_RIR1), SEQ ID NO: 522 (Pab_RIR1-3), SEQID NO: 523 (Pfu_KlbA), SEQ ID NO: 524 (Pho_IF2), SEQ ID NO: 525 (Pho_r-Gyr), SEQ ID NO: 526 (Pno_RPA2), SEQ ID NO: 527 (SaP-SETP3_helicase), SEQ ID NO: 528 (StP-Twort_ORF6), SEQ ID NO: 529 (Ter_DnaE-2), SEQ ID NO: 530 (Ter_RIR1-3), SEQ ID NO: 531 (Tko_helicase), SEQ ID NO: 532 (Tko_Pol-2_Pko_Pol-2-), SEQ ID NO: 533 (Tvo_VMA), SEQ ID NO: 534 (Tvu_DnaE-n_NC-terminus), SEQ ID NO: 535 (Unc-ERS_RIR1), SEQ ID NO: 536 (Tko_Pol-2_Pko_Pol-2-), SEQ ID NO: 537 (Tvo_VMA), SEQ ID NO: 538 (Tvu_DnaE-n_NC-terminus), SEQ ID NO: 539 (Unc-ERS_RIR2), SEQ ID NO: 540 (Tko_Pol-2_Pko_Pol-2-), SEQ ID NO: 541 (Tko_Pol-2_Pko_Pol-2-), SEQ ID NO: The reference sequences of NO:536 (synthetic construct Unc-ERS_RIR1_var7), SEQ ID NO:684 (mVMA:P77Cd) and SEQ ID NO:685 (mTth:P77Cd) have at least 70%, 72%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

In one embodiment, the intein-modified protease can comprise, consist essentially of, or consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 25 (Savinase-S114: VMA), SEQ ID NO: 26 (Savinase-T148: VMA), SEQ ID NO: 27 (Savinase-S166: VMA), SEQ ID NO: 28 (Savinase-S253: VMA), SEQ ID NO: 29 (Savinase-S269: VMA), SEQ ID NO: 30 (Savinase-S347: VMA), SEQ ID NO: 31 (Savinase-S114: Tth), SEQ ID NO: 32 (Savinase-T148: Tth), SEQ ID NO: 33 (Savinase-S166: Tth), SEQ ID NO: 34 (Savinase-S253: Tth), SEQ ID NO: 35 (Savinase-S269: Tth), SEQ ID NO: 36 (Savinase-S347: VMA), SEQ ID NO: 37 (Savinase-S347: VMA), SEQ ID NO: 38 (Savinase-S347: VMA), SEQ ID NO: 39 (Savinase-S403: VMA), SEQ ID NO: 40 (Savinase-S404: VMA), SEQ ID NO: 41 (Savinase-S405: VMA), SEQ ID NO: ID NO: 36 (Savinase-S347:Tth), SEQ ID NO: 120 (ProSavinase S46-mTth: EU59), SEQ ID NO: 121 (ProSavinase S62-mTth: EU59), SEQ ID NO: 122 (ProSavinase T77-mTth: EU59), SEQ ID NO: 123 (ProSavinase S86-mTth: EU59), SEQ ID NO: 124 (ProSavinase S100-mTth: EU59), SEQ ID NO: 125 ProSavinaseT109-mTth: EU59, SEQ ID NO: 126 (ProSavinase S135-mTth: EU59), SEQ ID NO: 127 (ProSavinase T148-mTth: EU59), SEQ ID NO: SEQ ID NO: 128 (ProSavinase S166-mTth: EU59), SEQ ID NO: 129 (ProSavinase T167-mTth: EU59), SEQ ID NO: 130 (ProSavinaseS196-mTth: EU59), SEQ ID NO: 131 (ProSavinase S208-mTth: EU59), SEQ ID NO: 132 (ProSavinase S239-mTth: EU59), SEQ ID NO: 133 (ProSavinase T243-mTth: EU59), SEQ ID NO: 134 (ProSavinase S269-mTth: EU59), SEQ ID NO: 135 (ProSavinase T285-mTth: EU59), SEQ ID NO: 136 (ProSavinase S293-mTth: EU59), SEQ ID NO: 137 (ProSavinase S317-mTth:EU59), SEQ ID NO:138(ProSavinase T318-mTth:EU59), SEQ ID NO:139 (ProSavinase T329-mTth:EU59), SEQ ID NO:140 (ProSavinase_S135:1:Aae_RIR2), SEQ ID NO:141 (ProSavinase_S135:2:Ace_RIR1), SEQ ID NO:SEQ ID NO:142(ProSavinase_S135:3:Aeh_DnaB-2), SEQ ID NO:143(ProSavinase_S135:4:Ani-FGSCA4_PRP8), SEQ ID NO:144(ProSavinase_S135:5:Ape_APE0745), SEQ ID NO:145(ProSavinase_S135:6:Avin_RIR1_BIL), SEQ ID NO:146(ProSavinase_S135:7:Bde-JEL197_RPB2), SEQ ID NO:147(ProSavinase_S135:8:Bde-JEL423_eIF-5), SEQ ID NO:148(ProSavinase_S135:9:BsuP-M1918_RIR1), SEQ ID NO:149(ProSavinase_S135:10:Cag_RIR1), SEQ ID NO:150(ProSavinase_S135:11:Cau_SpoVR), SEQ ID NO:151(ProSavinase_S135:12:Cbu_DnaB), SEQ ID NO:152(ProSavinase_S135:13:Ceu_ClpP), SEQ ID NO:153(ProSavinase_S135:14:Chy_RIR1), SEQ ID NO:154(ProSavinase_S135:15:Cth_ATPase_BIL), SEQ ID NO:155(ProSavinase_S135:16:Cth_TerA), SEQ ID NO:156(ProSavinase_S135:17:CV-NY2A_RIR1), SEQ ID NO:157(ProSavinase_S135:18:Cwa_PEP), SEQ ID NO:158(ProSavinase_S135:19:Cwa_RIR1), SEQ ID NO:159(ProSavinase_S135:20:Dhan_GLT1), SEQ ID NO:160(ProSavinase_S135:21:Fsp-CcI3_RIR1), SEQ ID NO:161(ProSavinase_S135:22:Gob_DnaE), SEQ ID NO:162(ProSavinase_S135:23:Gob_Hyp), SEQ ID NO:163(ProSavinase_S135:24:Gvi_RIR1-1), SEQ ID NO:164(ProSavinase_S135:25:Hhal_DnaB-1), SEQ ID NO:165(ProSavinase_S135:26:Hma_CDC21), SEQ ID NO:166(ProSavinase_S135:27:Hma_TopA), SEQ ID NO:167(ProSavinase_S135:28:Hsa-NRC1_CDC21), SEQ ID NO:168(ProSavinase_S135:29:Hvo_PolB), SEQ ID NO:169(ProSavinase_S135:30:Hwa_GyrB), SEQ ID NO:170(ProSavinase_S135:31:Hwa_MCM-1), SEQ ID NO:171(ProSavinase_S135:32:Hwa_MCM-4), SEQ ID NO:172(ProSavinase_S135:33:Hwa_PolB-2), SEQ ID NO:173(ProSavinase_S135:34:Hwa_Pol-II-1), SEQ ID NO:174(ProSavinase_S135:35:Hwa_Pol-II-2), SEQ ID NO:175(ProSavinase_S135:36:Hwa_RIR1-1), SEQ ID NO:176(ProSavinase_S135:37:Hwa_RIR1-2), SEQ ID NO:177(ProSavinase_S135:38:Hwa_rPol_App), SEQ ID NO: 178 (ProSavinase_S135:39: Kra_DnaB), SEQ ID NO: 179 (ProSavinase_S135:40: Mca_RIR1), SEQ ID NO: 180 (ProSavinase_S135:41; Memar_Pol-II), SEQ ID NO: 181 (ProSavinase_S135:42:Mex_helicase), SEQ ID NO: 182 (ProSavinase_S135:43:Mhu_Pol-II), SEQ ID NO: 183 (ProSavinase_S135:44:Mja_GF-6P), SEQ ID NO: 184 (ProSavinase_S135:45:Mja_helicase), SEQ ID NO: 185 (ProSavinase_S135:46; Mja_Hyp-1), SEQ ID NO: 186 (ProSavinase_S135:47:Mja_IF2), SEQ ID NO: 187 (ProSavinase_S135:48; Mja_Klba), SEQ ID NO: 188 (ProSavinase_S135:49:Mja_PEP), SEQ ID NO:189(ProSavinase_S135:50:Mja_Pol-1), SEQ ID NO:190(ProSavinase_S135:51:Mja_Pol-2), SEQ ID NO:191(ProSavinase_S135:52:Mja_RFC-1), SEQ ID NO:192(ProSavinase_S135:53;Mja_RFC-2), SEQ ID NO:193(ProSavinase_S135:54:Mja_RFC-3), SEQ ID NO:194(ProSavinase_S135:55:Mja_r-Gyr), SEQ ID NO:195(ProSavinase_S135:56:Mja_RNR-1), SEQ ID NO:196(ProSavinase_S135:57:Mja_RNR-2), SEQ ID NO:197(ProSavinase_S135:58:Mja_rPol_Ap), SEQ ID NO:198(ProSavinase_S135:59:Mja_rPol_App), SEQ ID NO:199(ProSavinase_S135:60:Mja_RtcB_Mja_Hyp-2), SEQ ID NO:200(ProSavinase_S135:61:Mja_TFIIB), SEQ ID NO:201(ProSavinase_S135:62:Mja_UDP_GD), SEQ ID NO:202(ProSavinase_S135:63:Mka_CDC48), SEQ ID NO:203(ProSavinase_S135:64:Mka_EF2), SEQ ID NO:204(ProSavinase_S135:65:Mka_RFC), SEQ ID NO:205(ProSavinase_S135:66:Mka_RtcB), SEQ ID NO:206(ProSavinase_S135:67:Mka_VatB), SEQ ID NO:207(ProSavinase_S135:68:MP-Be_gp51), SEQ ID NO:208(ProSavinase_S135:69; MP-Catera_gp206), SEQ ID NO:209(ProSavinase_S135:70:Mxa_RAD25), SEQ ID NO:210(ProSavinase_S135:71:Nfa_DnaB), SEQ ID NO:211(ProSavinase_S135:72:Nfa_Nfa15250), SEQ ID NO:212(ProSavinase_S135:73:Nfa_RIR1), SEQ ID NO:213(ProSavinase_S135:74:Nph_CDC21), SEQ ID NO:214(ProSavinase_S135:75:Nph_rPol_App), SEQ ID NO:215(ProSavinase_S135:76:Npu_GyrB), SEQ ID NO:216(ProSavinase_S135:77:Nsp-JS614_DnaB), SEQ ID NO:217(ProSavinase_S135:78:Nsp-PCC7120_RIR1), SEQ ID NO:218(ProSavinase_S135:79:Pab_CDC21-1), SEQ ID NO:219(ProSavinase_S135:80:Pab_CDC21-2), SEQ ID NO:220(ProSavinase_S135:81:Pab_IF2), SEQ ID NO: 221 (ProSavinase_S135:82:Pab_KlbA), SEQ ID NO: 222 (ProSavinase_S135:83:Pab_Lon), SEQ ID NO: 223 (ProSavinase_S135:84:Pab_Moaa), SEQ ID NO:224(ProSavinase_S135:85:Pab_Pol-II), SEQ ID NO:225(ProSavinase_S135:86:Pab_RFC-1), SEQ ID NO:226(ProSavinase_S135:87:Pab_RFC-2), SEQ ID NO:227(ProSavinase_S135:88:Pab_RIR1-1), SEQ ID NO:228(ProSavinase_S135:89:Pab_RIR1-2), SEQ ID NO:229(ProSavinase_S135:90:Pab_RIR1-3), SEQ ID NO:230(ProSavinase_S135:91:Pab_RtcB_Pab_Hyp-2), SEQ ID NO:231(ProSavinase_S135:92:Pab_VMA), SEQ ID NO:232(ProSavinase_S135:93:Pan_CHS2), SEQ ID NO:233(ProSavinase_S135:94:Pbr_PRP8), SEQ ID NO:234(ProSavinase_S135:95:Pch_PRP8), SEQ ID NO:235(ProSavinase_S135:96:Pfu_CDC21), SEQ ID NO:236(ProSavinase_S135:97:Pfu_IF2), SEQ ID NO: 237 (ProSavinase_S135:98; Pfu_KlbA), SEQ ID NO: 238 (ProSavinase_S135:99: Pfu_Lon), SEQ ID NO: 239 (ProSavinase_S135:100: Pfu_RFC), SEQ ID NO:240(ProSavinase_S135:101:Pfu_TopA), SEQ ID NO:241(ProSavinase_S135:102:Pho_CDC21-2), SEQ ID NO:242(ProSavinase_S135:103:Pho_IF2), SEQ ID NO:243(ProSavinase_S135:104:Pho_LHR), SEQ ID NO:244(ProSavinase_S135:105:Pho_Lon), SEQ ID NO:245(ProSavinase_S135:106:Pho_Pol_I), SEQ ID NO:246(ProSavinase_S135:107:Pho_RadA), SEQ ID NO:247(ProSavinase_S135:108:Pho_r-Gyr), SEQ ID NO:248(ProSavinase_S135:109:Pho_RtcB_Pho_Hyp-2), SEQ ID NO:249(ProSavinase_S135:110:Pho_VMA), SEQ ID NO:250(ProSavinase_S135:111:Pna_RIR1), SEQ ID NO:251(ProSavinase_S135:112:Pno_RPA2), SEQ ID NO:252(ProSavinase_S135:113:Posp-JS666_RIR1), SEQ ID NO:253(ProSavinase_S135:114:PP-PhiEL_ORF39), SEQ ID NO:254(ProSavinase_S135:115:Pst_VMA), SEQ ID NO:255(ProSavinase_S135:116:Rma_DnaB), SEQ ID NO: 256 (ProSavinase_S135:117:Rsp_Rir1), SEQ ID NO: 257 (ProSavinase_S135:118:SaP-SETP3_helicase), SEQ ID NO: 258 (ProSavinase_S135:119:Sav_helicase), SEQ ID NO: 259 (ProSavinase_S135:120:Sex-IFO1128_VMA), SEQ ID NO: 260 (ProSavinase_S135:121:Smar_1471), SEQ ID NO:261(ProSavinase_S135:122:Smar_MCM2), SEQ ID NO:262(ProSavinase_S135:123:Sru_DnaB), SEQ ID NO:263(ProSavinase_S135:124:Sru_PolBc), SEQ ID NO:264(ProSavinase_S135:125:Ssp_DnaB), SEQ ID NO:265(ProSavinase_S135:126:Ssp_GyrB), SEQ ID NO:266(ProSavinase_S135:127:StP-Twort_ORF6), SEQ ID NO:267(ProSavinase_S135:128:Tag_Pol-1_Tsp-TY_Pol-1), SEQ ID NO:268(ProSavinase_S135:129:Tag_Pol-2_Tsp-TY_Pol-2_T134), SEQ ID NO:269(ProSavinase_S135:130:Ter_DnaB-1Ter_DnaB-1), SEQ ID NO:270(ProSavinase_S135:131:Ter_DnaE-2), SEQ ID NO:271(ProSavinase_S135:132:Ter_DnaE-3nc_NC-terminal), SEQID NO:272(ProSavinase_S135:133:Ter_Ndse-2), SEQ ID NO:273(ProSavinase_S135:134:Ter_RIR1-3Ter_RIR1-3), SEQ ID NO:274(ProSavinase_S135:135:Ter_RIR1-4), SEQID NO:275(ProSavinase_S135:136:Ter_Snf2), SEQ ID NO:276(ProSavinase_S135:137:Tfu_Pol-2), SEQ ID NO:277(ProSavinase_S135:138:Tfus_RecA-1), SEQ ID NO:278(ProSavinase_S135:139:Tfus_RecA-2), SEQ ID NO:279 (ProSavinase_S135:140:Thy_Pol-1), SEQ ID NO:280 (ProSavinase_S135:141:Tko_CDC21-2), SEQ ID NO:281 (ProSavinase_S135:142:Tko_helicase), SEQ ID NO:282(ProSavinase_S135:143:Tko_IF2), SEQ ID NO:283(ProSavinase_S135:144:Tko_LHR), SEQ ID NO:284(ProSavinase_S135:145:Tko_Pol-2_Pko_Pol-2), SEQ ID NO:285(ProSavinase_S135:146:Tko_RadA), SEQ ID NO:286(ProSavinase_S135:147:Tko_r-Gyr), SEQ ID NO:287(ProSavinase_S135:148:Tko_RIR1-1), SEQ ID NO:288(ProSavinase_S135:149:Tko_TopA), SEQ ID NO:289(ProSavinase_S135:150:Tth-HB27_DnaE-2), SEQ ID NO:290(ProSavinase_S135:151:Tth-HB27_RIR1-1), SEQ ID NO:291(ProSavinase_S135:152:Tth-HB27_RIR1-2), SEQ ID NO:292(ProSavinase_S135:153:Tvo_VMA), SEQ ID NO:293(ProSavinase_S135:154:Tvu_DnaE-n_NC-terminal), SEQ ID NO:294(ProSavinase_S135:155:Unc-ERS_RIR1), SEQ ID NO:295(ProSavinase_S135:156:Zba_VMA), SEQ ID NO:296(ProSavinase_S135:157:Zro_VMAZro_VMA), SEQ ID NO:297(ProSavinase_S317:1:Aae_RIR2), SEQ ID NO:298(ProSavinase_S317:2:Ace_RIR1), SEQ ID NO:299(ProSavinase_S317:3:Aeh_DnaB-2), SEQ ID NO:300(ProSavinase_S317:4:Ani-FGSCA4_PRP8), SEQ ID NO:301(ProSavinase_S317:5:Ape_APE0745), SEQ ID NO:302(ProSavinase_S317:6:Avin_RIR1_BIL), SEQ ID NO:303(ProSavinase_S317:7:Bde-JEL197_RPB2), SEQ ID NO:304(ProSavinase_S317:8:Bde-JEL423_eIF-5B), SEQ ID NO:305(ProSavinase_S317:9:BsuP-M1918_RIR1), SEQ ID NO:306(ProSavinase_S317:10:Cag_RIR1), SEQ ID NO:307(ProSavinase_S317:11:Cau_SpoVR), SEQ ID NO:308(ProSavinase_S317:12:Cbu_DnaB), SEQ ID NO:309(ProSavinase_S317:13:Ceu_ClpP), SEQ ID NO:310(ProSavinase_S317:14:Chy_RIR1), SEQ ID NO:311(ProSavinase_S317:15:Cth_ATPase_BIL), SEQ ID NO:312(ProSavinase_S317:16:Cth_TerA), SEQ ID NO:313(ProSavinase_S317:17:CV-NY2A_RIR1), SEQ ID NO:314(ProSavinase_S317:18:Cwa_PEP), SEQ ID NO:315(ProSavinase_S317:19:Cwa_RIR1), SEQID NO:316(ProSavinase_S317:20:Dhan_GLT1), SEQ ID NO:317(ProSavinase_S317:21:Fsp-CcI3_RIR1), SEQ ID NO:318(ProSavinase_S317:22:Gob_DnaE), SEQ ID NO:319(ProSavinase_S317:23:Gob_Hyp), SEQ ID NO:320(ProSavinase_S317:24:Gvi_RIR1-1), SEQ ID NO:321(ProSavinase_S317:25:Hhal_DnaB-1), SEQ ID NO:322(ProSavinase:S317:26:Hma_CDC21), SEQ ID NO:323(ProSavinase_S317:27:Hma_TopA), SEQ ID NO:324(ProSavinase_S317:28:Hsa-NRC1_CDC21), SEQ ID NO:325(ProSavinase_S317:29:Hvo_PolB), SEQ ID NO:326(ProSavinase_S317:30:Hwa_GyrB), SEQ ID NO:327(ProSavinase_S317:31:Hwa_MCM-1), SEQ ID NO:328(ProSavinase_S317:32:Hwa_MCM-4), SEQ ID NO:329(ProSavinase_S317:33:Hwa_PolB-2), SEQ ID NO:330(ProSavinase_S317:34:Hwa_Pol-II-1), SEQ ID NO:331(ProSavinase_S317:35:Hwa_Pol-II-2), SEQ ID NO:332(ProSavinase_S317:36:Hwa_RIR1-1), SEQ ID NO:333(ProSavinase_S317:37:Hwa_RIR1-2), SEQ ID NO:334(ProSavinase_S317:38:Hwa_rPol_App), SEQ ID NO:335(ProSavinase_S317:39:Kra_DnaB), SEQ ID NO:336(ProSavinase_S317:40:Mca_RIR1), SEQ ID NO: 337 (ProSavinase_S317:41: Memar_Pol-II), SEQ ID NO: 338 (ProSavinase_S317:42: Mex_helicase), SEQ ID NO: 339 (ProSavinase_S317:43: Mhu_Pol-II), SEQ ID NO: 340 (ProSavinase_S317:44:Mja_GF-6P), SEQ ID NO: 341 (ProSavinase_S317:45:Mja_helicase), SEQ ID NO:342(ProSavinase_S317:46:Mja_Hyp-1), SEQ ID NO:343(ProSavinase_S317:47:Mja_IF2), SEQ ID NO:344(ProSavinase_S317:48:Mja_Klba), SEQ ID NO:345(ProSavinase_S317:49:Mja_PEP), SEQ ID NO:346(ProSavinase_S317:50:Mja_Pol-1), SEQ ID NO:347(ProSavinase_S317:51:Mja_Pol-2), SEQ ID NO:348(ProSavinase_S317:52:Mja_RFC-1), SEQ ID NO:349(ProSavinase_S317:53:Mja_RFC-2), SEQ ID NO:350(ProSavinase:S317:54:Mja_RFC-3), SEQ ID NO:351(ProSavinase_S317:55:Mja_r-Gyr), SEQ ID NO:352(ProSavinase_S317:56:Mja_RNR-1), SEQ ID NO:353(ProSavinase_S317:57:Mja_RNR-2), SEQ ID NO:354(ProSavinase_S317:58:Mja_rPol_Ap), SEQ ID NO:355(ProSavinase_S317:59:Mja_rPol_App), SEQ ID NO:356(ProSavinase_S317:60:Mja_RtcB_Mja_Hyp-2), SEQ ID NO:357(ProSavinase_S317:61:Mja_TFIIB), SEQ ID NO:358(ProSavinase_S317:62:Mja_UDP_GD), SEQ ID NO:359(ProSavinase_S317:63:Mka_CDC48), SEQ ID NO:360(ProSavinase_S317:64:Mka_EF2), SEQ ID NO: 361 (ProSavinase_S317:65:Mka_RFC), SEQ ID NO: 362 (ProSavinase_S317:66; Mka_RtcB), SEQ ID NO: 363 (ProSavinase_S317:67:Mka_VatB), SEQ ID NO:364(ProSavinase_S317:68:MP-Be_gp51), SEQ ID NO:365(ProSavinase_S317:69:MP-Catera_gp206), SEQ ID NO:366(ProSavinase_S317:70:Mxa_RAD25), SEQ ID NO:367(ProSavinase_S317:71:Nfa_DnaB), SEQ ID NO:368(ProSavinase_S317:72:Nfa_Nfa15250), SEQ ID NO:369(ProSavinase_S317:73:Nfa_RIR1), SEQ ID NO:370(ProSavinase_S317:74:Nph_CDC21), SEQ ID NO:371(ProSavinase_S317:75:Nph_rPol_App), SEQ ID NO:372(ProSavinase_S317:76:Npu_GyrB), SEQ ID NO:373(ProSavinase_S317:77:Nsp-JS614_DnaB), SEQ ID NO:374(ProSavinase_S317:78:Nsp-PCC7120_RIR1), SEQ ID NO:375(ProSavinase_S317:79:Pab_CDC21-1), SEQ ID NO:376(ProSavinase_S317:80:Pab_CDC21-2), SEQ ID NO:377(ProSavinase_S317:81:Pab_IF2), SEQ ID NO:378(ProSavinase_S317:82:Pab_KlbA), SEQ ID NO:379(ProSavinase_S317:83:Pab_Lon), SEQ ID NO:380(ProSavinase_S317:84:Pab_Moaa), SEQ ID NO:381(ProSavinase_S317:85:Pab_Pol-II), SEQ ID NO:382(ProSavinase_S317:86:Pab_RFC-1), SEQ ID NO:S 383(ProSavinase_S317:87:Pab_RFC-2), SEQ ID NO:384(ProSavinase_S317:88:Pab_RIR1-1), SEQ ID NO:385(ProSavinase_S317:89:Pab_RIR1-2), SEQ ID NO:386(ProSavinase_S317:90:Pab_RIR1-3), SEQ ID NO:387(ProSavinase_S317:91:Pab_RtcB_Pab_Hyp-2), SEQ ID NO:388(ProSavinase_S317:92:Pab_VMA), SEQ ID NO:389(ProSavinase_S317:93:Pan_CHS2), SEQ ID NO:390(ProSavinase_S317:94:Pbr_PRP8), SEQ ID NO:391(ProSavinase_S317:95:Pch_PRP8), SEQ ID NO:392(ProSavinase_S317:96:Pfu_CDC21), SEQ ID NO:393(ProSavinase_S317:97:Pfu_IF2), SEQ ID NO:394(ProSavinase_S317:98:Pfu_KlbA), SEQ ID NO:395(ProSavinase_S317:99:Pfu_Lon), SEQ ID NO:396(ProSavinase_S317:100:Pfu_RFC), SEQ ID NO:397(ProSavinase_S317:101:Pfu_TopA), SEQ ID NO:398(ProSavinase_S317:102:Pho_CDC21-2), SEQ ID NO:399(ProSavinase_S317:103:Pho_IF2), SEQ ID NO:400(ProSavinase_S317:104:Pho_LHRPho_LHR), SEQ ID NO:401(ProSavinase_S317:105:Pho_Lon), SEQ ID NO:402(ProSavinase_S317:106:Pho_Pol_I), SEQ ID NO:403(ProSavinase_S317:107:Pho_RadA), SEQ ID NO:404(ProSavinase_S317:108:Pho_r-Gyr)、SEQ ID NO:405(ProSavinase_S317:109:Pho_RtcB_Pho_Hyp-2), SEQ ID NO:406(ProSavinase_S317:110:Pho_VMA), SEQ ID NO:407(ProSavinase_S317:111:Pna_RIR1), SEQ ID NO:408(ProSavinase_S317:112:Pno_RPA2), SEQ ID NO:409(ProSavinase_S317:113:Posp-JS666_RIR1), SEQ ID NO:410(ProSavinase_S317:114:PP-PhiEL_ORF39), SEQ ID NO:411(ProSavinase_S317:115:Pst_VMA), SEQ ID NO:412(ProSavinase_S317:116:Rma_DnaB), SEQ ID NO:413(ProSavinase_S317:117:Rsp_Rir1), SEQ ID NO:414(ProSavinase_S317:118:SaP-SETP3_helicase), SEQID NO: 415 (ProSavinase_S317:119:Sav_helicase), SEQ ID NO: 416 (ProSavinase_S317:120:Sex-IFO1128_VMA), SEQ ID NO: 417 (ProSavinase_S317:121:Smar_1471), SEQ ID NO:418(ProSavinase_S317:122:Smar_MCM2), SEQ ID NO:419(ProSavinase_S317:123:Sru_DnaB), SEQ ID NO:420(ProSavinase_S317:124:Sru_PolBc), SEQ ID NO:421(ProSavinase_S317:125:Ssp_DnaB), SEQ ID NO:422(ProSavinase_S317:126:Ssp_GyrB), SEQ ID NO:423(ProSavinase_S317:127:StP-Twort_ORF6), SEQ ID NO:424(ProSavinase_S317:128:Tag_Pol-1_Tsp-TY_Pol-1), SEQ ID NO:425(ProSavinase_S317:129:Tag_Pol-2_Tsp-TY_Pol-2_T134), SEQ ID NO:426(ProSavinase_S317:130:Ter_DnaB-1), SEQ ID NO: 427 (ProSavinase_S317:131: Ter_DnaE-2), SEQ ID NO: 428 (ProSavinase_S317:132: Ter_DnaE-3nc_NC-terminal), SEQ ID NO: 429 (ProSavinase_S317:133: Ter_Ndse-2), SEQ ID NO:430(ProSavinase_S317:134:Ter_RIR1-3), SEQ ID NO:431(ProSavinase_S317:135:Ter_RIR1-4), SEQ ID NO:432(ProSavinase_S317:136:Ter_Snf2), SEQ ID NO:433(ProSavinase_S317:137:Tfu_Pol-2), SEQ ID NO:434(ProSavinase_S317:138:Tfus_RecA-1), SEQ ID NO:435(ProSavinase_S317:139:Tfus_RecA-2), SEQ ID NO:436(ProSavinase_S317:140:Thy_Pol-1), SEQ ID NO: 437 (ProSavinase_S317:141:Tko_CDC21-2), SEQ ID NO: 438 (ProSavinase_S317:142:Tko_helicase), SEQ ID NO: 439 (ProSavinase_S317:143:Tko_IF2), SEQ ID NO:440(ProSavinase_S317:144:Tko_LHR), SEQ ID NO:441(ProSavinase_S317:145:Tko_Pol-2_Pko_Pol-2), SEQ ID NO:442(ProSavinase_S317:146:Tko_RadA), SEQ ID NO:443(ProSavinase_S317:147:Tko_r-Gyr), SEQ ID NO:444(ProSavinase_S317:148:Tko_RIR1-1), SEQ ID NO:445(ProSavinase_S317:149:Tko_TopA), SEQ ID NO:446(ProSavinase_S317:150:Tth-HB27_DnaE-2), SEQ ID NO:447(ProSavinase_S317:151:Tth-HB27_RIR1-1), SEQ ID NO:448(ProSavinase_S317:152Tth-HB27_RIR1-2), SEQ ID NO: 449 (ProSavinase_S317:153:Tvo_VMA), SEQ ID NO: 450 (ProSavinase_S317:154:Tvu_DnaE-n_NC-terminal), SEQ ID NO: 451 (ProSavinase_S317:155:Unc-ERS_RIR1), SEQ ID NO:452(ProSavinase_S317:156:Zba_VMA), SEQ ID NO:453(ProSavinase_S317:157:Zro_VMA), SEQ ID NO:496(ProSavinase_S317:155_var7), SEQ ID NO:686(iproSavS135:mVMA:P77Cd), SEQ ID NO:687(iproSavS265:mVMA:P77Cd), SEQ ID NO: 688 (iproSavS269:mVMA:P77Cd), SEQ ID NO: 689 (iproSavS293:mVMA:P77Cd), SEQ ID NO: 690 (iproSavS312:mVMA:P77Cd), SEQ ID NO: 691 (iproSavS317:mVMA:P77Cd), SEQ ID NO:692 (iproSavS326:mVMA:P77Cd), SEQ ID NO:693 (iproSavS135:mTth:P77Cd), SEQ ID NO:694 (iproSavS269:mTth:P77Cd), SEQ ID NO:695 (iproSavS293:mTth:P77Cd) and SEQ ID The reference sequences in the group consisting of NO:696 (iproSavS317:mTth:P77Cd) have at least 70%, 72%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

Inteins can spontaneously splice intein-modified proteases. Inteins can inducibly cause cis-splicing of intein-modified proteases by exposure to inducing conditions as described herein.

One embodiment includes an expression cassette. The expression cassette can include a polynucleotide encoding an intein-modified protease. The intein-modified protease can be any of the ones described herein.

In one embodiment, the polynucleotide may include a sequence encoding any target protease. The polynucleotide may include a sequence encoding a keratinase. The polynucleotide may include a sequence encoding a savinase. The polynucleotide may include a sequence encoding a savinase. The polynucleotide may include a sequence having at least 70%, 72%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the reference sequence of SEQ ID NO: 41 (Q53521) or SEQ ID NO: 59 (P29600).

In one embodiment, the polynucleotide may comprise a sequence encoding an intein capable of trans-splicing of an intein-modified protease.The polynucleotide may comprise a sequence encoding an N-intein or a C-intein. The polynucleotide may comprise a sequence that is at least 70%, 72%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference sequence selected from the group consisting of SEQ ID NO: 42 (DnaE-N), SEQ ID NO: 43 (DnaE-C), SEQ ID NO: 674 (gp41-1-N), SEQ ID NO: 675 (gp41-1-C), SEQ ID NO: 676 (gp41-8-N), SEQ ID NO: 677 (gp41-8-C), SEQ ID NO: 678 (IMPDH-1-N), SEQ ID NO: 679 (IMPDH-1-C), SEQ ID NO: 680 (NrdJ-1-C), and SEQ ID NO: 681 (NrdJ-1-N).

In one embodiment, the polynucleotide may include a sequence encoding a first portion of an intein-modified protease. The first portion may include an N-extein of a target protease and an N-intein of an intein. The carboxyl terminus of the N-extein may be fused to the amino terminus of the N-intein. The polynucleotide may include a polypeptide selected from the group consisting of SEQ ID NO: 93 (Q53521-T108: DnaE-N), SEQ ID NO: 95 (S154: DnaE-N), SEQ ID NO: 97 (Q53521-S234: DnaE-N), SEQ ID NO: 99 (Q53521-S260: DnaE-N), SEQ ID NO: 101 (Q5321-S263-DnaE-N), SEQ ID NO: 103 (Q53521-T317: DnaE-N), SEQ ID NO: 587 (NI-GG-6H), SEQ ID NO: 589 (S135_IMPDH-NI), SEQ ID NO: 590 (S269_IMPDH-NI), SEQ ID NO: 591 (S293_IMPDH-NI), SEQ ID NO: 592 (S394_IMPDH-NI), SEQ ID NO: 593 (S406_IMPDH-NI), SEQ ID NO: 594 (S407_IMPDH-NI), SEQ ID NO: 595 (S408_IMPDH-NI), SEQ ID NO: 596 (S409_IMPDH-NI), SEQ ID NO: 597 (S410_IMPDH-NI), SEQ ID NO: 598 (S411_IMPDH-NI), SEQ ID NO: 599 (S421_IMPDH-NI), SEQ ID NO: 610 (S422_IMPDH-NI), SEQ ID NO: 611 (S423_IMPDH-NI), SEQ ID NO: 612 (S424_IMPDH-NI), SEQ ID NO: 613 (S425_IMPDH-NI), SEQ ID NO: NO: 592 (S317_IMPDH-NI), SEQ ID NO: 593 (T318_IMPDH-NI), SEQ ID NO: 594 (S135_gp41-1-NI), SEQ ID NO: 595 (S269_gp41-1-NI), SEQ ID NO: 596 (S293_gp41-1-NI), SEQ ID NO: 597 (S317_gp41-1-NI), SEQ ID NO: 598 (T318_gp41-1-NI), SEQ ID NO: 599 (S135_gp41-8-NI), SEQ ID NO: 600 (S269_gp41-8-NI), SEQ ID NO: 601 (S293_gp41-8-NI), SEQ ID NO: 602 (S317_gp41-8-NI), SEQ ID The reference sequences in the group consisting of SEQ ID NO: 603 (T318_gp41-8-NI), SEQ ID NO: 604 (S135_NrdJ-1-NI), SEQ ID NO: 605 (S269_NrdJ-1-NI), SEQ ID NO: 606 (S293_NrdJ-1-NI), SEQ ID NO: 607 (S317_NrdJ-1-NI) and SEQ ID NO: 608 (T318_NrdJ-1-NI) have sequences that are at least 70%, 72%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical.

The polynucleotide may include a sequence encoding a second portion of an intein-modified protease. The second portion may include a C-intein of the intein and a C-extein of the target protease. The carboxyl terminus of the C-intein may be fused to the amino terminus of the C-extein. The polynucleotide may comprise a polypeptide selected from the group consisting of SEQ ID NO: 94 (DnaE-C: T108-Q53521-C), SEQ ID NO: 96 (DnaE-C: S154-Q53521-C), SEQ ID NO: 98 (DnaE-C: S234-Q53521-C), SEQ ID NO: 100 (DnaE-C: S260-Q53521-C), SEQ ID NO: 102 (DnaE-C: S263-Q53521-C), SEQ ID NO: 104 (DnaE-C: T317-Q53521-C), SEQ ID NO: 588 (IC-SUMO-6H), SEQ ID NO: 609 (S135_IMPDH-IC), SEQ ID NO: 610 (S269_IMPDH-IC), SEQ ID NO: 621 (S280-Q53521-C), SEQ ID NO: 622 (S281-Q53521-C), SEQ ID NO: 623 (S282-Q53521-C), SEQ ID NO: 624 (S283-Q53521-C), SEQ ID NO: 625 (S284-Q53521-C), SEQ ID NO: 626 (S285-Q53521-C), SEQ ID NO: 627 (S286-Q53521-C), SEQ ID NO: 628 (S287-Q53521-C), NO: 611 (S293_IMPDH-IC), SEQ ID NO: 612 (S317_IMPDH-IC), SEQ ID NO: 613 (T318_IMPDH-IC), SEQ ID NO: 614 (S135_gp41-1-IC), SEQ ID NO: 615 (S269_gp41-1-IC), SEQ ID NO: 616 (S293_gp41-1-IC), SEQ ID NO: 617 (S317_gp41-1-IC), SEQ ID NO: 618 (T318_gp41-1-IC), SEQ ID NO: 619 (S135_gp41-8-IC), SEQ ID NO: 620 (S269_gp41-8-IC), SEQ ID NO: 621 (S293_gp41-8-IC), SEQ ID The reference sequences in the group consisting of SEQ ID NO: 622 (S317_gp41-8-IC), SEQ ID NO: 623 (T318_gp41-8-IC), SEQ ID NO: 624 (S135_NrdJ-1-IC), SEQ ID NO: 625 (S269_NrdJ-1-IC), SEQ ID NO: 626 (S293_NrdJ-1-IC), SEQ ID NO: 627 (S317_NrdJ-1-IC) and SEQ ID NO: 628 (T318_NrdJ-1-IC) have sequences that are at least 70%, 72%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical.

The polynucleotides encoding the first and second parts of the intein-modified protease can be expressed separately. The first and second parts can be expressed in different compartments of the host cell. The first and second parts can also be expressed in different host lines.

In one embodiment, a polynucleotide may include a sequence encoding an intein that is capable of cis-splicing by a protease that modifies the intein. The polynucleotide may include a polypeptide selected from the group consisting of SEQ ID NO: 72 (mTth: EU59 intein), SEQ ID NO: 105 (VMA), SEQ ID NO: 106 (Tth), SEQ ID NO: 40 (Tth), SEQ ID NO: 119 (mTth: EU59 intein), SEQ ID NO: 634 (Cth_ATPase_BIL), SEQ ID NO: 635 (Cwa_RIR1), SEQ ID NO: 636 (Dhan_GLT1), SEQ ID NO: 637 (FSP-CcI3_RIR1), SEQ ID NO: 638 (Gob_Hyp), SEQ ID NO: 639 (Gvi_RIR1-1), SEQ ID NO: 640 (Hhal_DnaB-1), SEQ ID NO: 641 (Hma_CDC21), SEQ ID NO: 642 (Hwa_MCM-1), SEQ ID NO: 643 (Hwa_PolB-2), SEQ ID NO: 644 (Hwa_PolB-3), SEQ ID NO: 645 (Hwa_DnaB-4), SEQ ID NO: 646 (Hwa_DnaB-5), SEQ ID NO: 647 (Hwa_DnaB-6), SEQ ID NO: 648 (Hwa_DnaB-7), SEQ ID NO: 649 (Hwa_DnaB-8), SEQ ID NO: 650 (Hwa_DnaB-9), SEQ ID NO: 651 (Hwa_DnaB-1 NO: 644 (Hwa_RIR1-1), SEQ ID NO: 645 (Hwa_RIR1-2), SEQ ID NO: 646 (Hwa_rPol_App), SEQ ID NO: 647 (Kra_DnaB), SEQ ID NO: 648 (Mca_RIR1), SEQ ID NO: 649 (Memar_Pol-II), SEQ ID NO: 650 (Mex_Helicase), SEQ ID NO: 651 (Mhu_Pol-II), SEQ ID NO: 652 (Mja_Klba), SEQ ID NO: 653 (Mja_PEP), SEQ ID NO: 654 (Mja_Pol-2), SEQ ID NO: 655 (Mja_RFC-3), SEQ ID NO: 656 (Mja_r-Gyr), SEQ ID NO: 657 (MP-Be_gp51), SEQ ID NO: 658 (NSP-PCC7120_RIR1), SEQ ID NO: 659 (Pab_RIR1-3), SEQ ID NO: 660 (Pfu_KlbA), SEQ ID NO: 661 (Pho_IF2), SEQ ID NO: 662 (Pho_r-Gyr), SEQ ID NO: 663 (Pno_RPA2), SEQ ID NO: 664 (SAP-SETP3_Helicase), SEQ ID NO: 665 (STP-Twort_ORF6), SEQ ID NO: 666 (Ter_DnaE-2), SEQ ID NO: 667 (Ter_RIR1-3), SEQ ID NO: 668 (Tko_Helicase), SEQ ID NO: 669 (Tko_Pol-2_Pko_Pol-2), SEQ ID NO: 670 (Tvo_VMA), SEQ ID NO: 671 (Tvu_DnaE-n_NC-terminus), SEQ ID NO: 672 (UNC-ERS_RIR1), SEQ ID NO: 673 (UNC-ERS_RIR2), SEQ ID NO: 674 (UNC-ERS_RIR3), SEQ ID NO: 675 (UNC-ERS_RIR4), SEQ ID NO: 676 (UNC-ERS_RIR5), SEQ ID NO: 677 (UNC-ERS_RIR6), SEQ ID NO: 678 (UNC-ERS_RIR7), SEQ ID NO: 679 (UNC-ERS_RIR8), SEQ ID NO: 680 (UNC-ERS_RIR9), SEQ ID NO: 681 (UNC-ERS_RIR1), SEQ ID NO: The reference sequences in the group consisting of NO: 673 (synthetic construct Unc-ERS_RIR1_var7), SEQ ID NO: 699 (mVMA: P77Cd) and SEQ ID NO: 700 (mTth: P77Cd) have sequences with at least 70%, 72%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

In one embodiment, the polynucleotide comprises, consists essentially of, or consists of a sequence selected from the group consisting of SEQ ID NO: 107 (Savinase-S114: VMA), SEQ ID NO: 108 (Savinase-T148: VMA), SEQ ID NO: 109 (Savinase-S166: VMA), SEQ ID NO: 110 (Savinase-S253: VMA), SEQ ID NO: 111 (Savinase-S269: VMA), SEQ ID NO: 112 (Savinase-S347: VMA), SEQ ID NO: 113 (Savinase-S114: Tth), SEQ ID NO: 114 (Savinase-T148: Tth), SEQ ID NO: 115 (Savinase-S166: Tth), SEQ ID NO: 116 (Savinase-S253: Tth), SEQ ID NO: 117 (Savinase-S347: VMA), SEQ ID NO: 118 (Savinase-S347: VMA), SEQ ID NO: 119 (Savinase-S403: Tth), SEQ ID NO: 120 (Savinase-S404: Tth), SEQ ID NO: 121 (Savinase-S405: Tth), SEQ ID NO: 122 (Savinase-S406: Tth), SEQ ID NO: 123 (Savinase-S407: Tth), SEQ ID NO: 124 (Savinase-S408: Tth), SEQ ID NO: 125 NO: 117 (Savinase-S269: Tth), SEQ ID NO: 118 (Savinase-S347: Tth), SEQ ID NO: 73 (ProSavinaseS46-mTth: EU59), SEQ ID NO: 74 (ProSavinase S62-mTth: EU59), SEQ ID NO: 75 (ProSavinase T77-mTth: EU59), SEQ ID NO: 76 (ProSavinase S86-mTth: EU59), SEQ ID NO: 77 (ProSavinase S100-mTth: EU59), SEQ ID NO: 78 ProSavinase T109-mTth: EU59, SEQ ID NO: 79 (ProSavinase S135-mTth: EU59), SEQ ID NO: 80 (ProSavinase T148-mTth: EU59), SEQ ID NO: SEQ ID NO: 81 (ProSavinase S166-mTth: EU59), SEQ ID NO: 82 (ProSavinase T167-mTth: EU59), SEQ ID NO: 83 (ProSavinase S196-mTth: EU59), SEQ ID NO: 84 (ProSavinase S208-mTth: EU59), SEQ ID NO: 85 (ProSavinase S239-mTth: EU59), SEQ ID NO: 86 (ProSavinase T243-mTth: EU59), SEQ ID NO: 87 (ProSavinase S269-mTth: EU59), SEQ ID NO: 88 (ProSavinase T285-mTth: EU59), SEQ ID NO: 89 (ProSavinase S293-mTth: EU59), SEQ ID NO: 90 (ProSavinase S317-mTth: EU59), SEQ ID NO: 91 (ProSavinase_T318-mTth: EU59), SEQ ID NO: 92 (ProSavinase_T329-mTth: EU59), SEQ ID NO: 545 (ProSavinase_S135: 15: Cth_ATPase_BIL), SEQ ID NO: 546 (ProSavinase_S135: 38: Hwa_rPol_App), SEQ ID NO: 547 (ProSavinase_S135: 39: Kra_DnaB), SEQ ID NO: 548 (ProSavinase_S135: 48: Mja_Klba), SEQ ID NO: 549 (ProSavinase_S135: 54: Mja_Pol-2), SEQ ID NO: 550 (ProSavinase_S135: 54: Mja_RFC-3), SEQ ID NO: 551 (ProSavinase_S135: 55: Mja_r-GYR), SEQ ID NO: 552 (ProSavinase_S135: 142: Tko_helicase), SEQ ID NO: 553 (ProSavinase_S135: 145: Tko_Pol-2_Pko_Pol-2), SEQ ID NO: 554 (ProSavinase_S135: 153: Tvo_VMA), SEQ ID NO: 555 (ProSavinase_S135: 154: Tvu_DnaE-n_NC-terminal), SEQ ID NO: 556 (ProSavinase_S317: 19: Cwa_RIR1), SEQ ID NO: 557 (ProSavinase_S317: 20: Dhan_GLT1), SEQ ID NO: 558 (ProSavinase_S317: 21: FSP-CcI3_RIR1), SEQ ID NO: 559 (ProSavinase_S317: 23: Gob_Hyp), SEQ ID NO: 560 (ProSavinase_S317: 24: Gvi_RIR1-1), SEQ ID NO: 561 (ProSavinase_S317: 25: Hha1_DnaB-1), SEQ ID NO: 562 (ProSavinase_S317: 26: Hma_CDC21), SEQ ID NO: 563 (ProSavinase_S317: 31: Hwa_MCM-1), SEQ ID NO: 564 (ProSavinase_S317: 33: Hwa_PolB-2), SEQ ID NO: SEQ ID NO: 565 (ProSavinase_S317: 36: Hwa_RIR1-1), SEQ ID NO: 566 (ProSavinase_S317: 37: Hwa_RIR1-2), SEQ ID NO: 567 (ProSavinase_S317: 39: Kra_DnaB), SEQ ID NO: 568 (ProSavinase_S317: 40: Mca_RIR1), SEQ ID NO: 569 (ProSavinase_S317: 41: Memar_Pol-II), SEQ ID NO: 570 (ProSavinase_S317: 42: Mhu_Pol-II), SEQ ID NO: 571 (ProSavinase_S317: 43: Mhu_Pol-II), SEQ ID NO: 572 (ProSavinase_S317: 49: Mja_PEP), SEQ ID NO: 573 (ProSavinase_S317: 68: MP-Be_gp51), SEQ ID NO: 574 (ProSavinase_S317: 78: NSP-PCC7120_RIR1), SEQ ID NO: 575 (ProSavinase_S317: 90: Pab_RIR1-3), SEQ ID NO: 576 (ProSavinase_S317: 98: Pfu_KlbA), SEQ ID NO: 577 (ProSavinase_S317: 103: Pho_IF2), SEQ ID NO: 578 (ProSavinase_S317: 108: Pho_r-Gyr), SEQ ID NO: 579 (ProSavinase_S317: 112: Pno_RPA2), SEQ ID NO: 580 (ProSavinase_S317: 118: SAP-SETP3_Helicase), SEQ ID NO: 581 (ProSavinase_S317: 127: STP Twort_ORF6), SEQ ID NO: 582 (ProSavinase_S317: 131: Ter_DnaE-2), SEQ ID NO: 583 (ProSavinase_S317: 134: Ter_RIR1-3), SEQ ID NO: 584 (ProSavinase_S317: 142: Tko_helicase), SEQ ID NO: 585 (ProSavinase_S317: 145: Tko_Pol-2_Pko_Pol-2), SEQ ID NO: 586 (ProSavinase_S317: 155: Unc-ERS_RIR1), SEQ ID NO: 701 (iproSavS135: mVMA: P77Cd), SEQ ID NO: 702 (iproSavS265: mVMA: P77Cd), SEQ ID NO: 703 (iproSavS269: mVMA: P77Cd), SEQ ID NO: 704 (iproSavS293: mVMA: P77Cd), SEQ ID NO: 705 (iproSavS312: mVMA: P77Cd), SEQ ID NO: 706 (iproSavS317: mVMA: P77Cd), SEQ ID NO: 707 (iproSavS326: mVMA: P77Cd), SEQ ID NO: 708 (iproSavS135: mTth: P77Cd), SEQ ID NO: 709 (iproSavS269: mTth: P77Cd), SEQ ID NO: 710 (iproSavS293: mTth: P77Cd) and SEQ ID The reference sequences in the group consisting of NO:711 (iproSavS317:mTth:P77Cd) have at least 70%, 72%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

The polynucleotide sequences, isolated nucleic acids, vectors, or any other DNA constructs described herein or utilized in the methods herein may be operably linked to one or more regulatory elements. Such regulatory elements may be promoters. The promoter may be a constitutive promoter, which provides for transcription of the polynucleotide sequence in most cells, tissues, and organs throughout a plant, and at many, but not necessarily all, stages of development. The promoter may be an inducible promoter, which initiates transcription of the polynucleotide sequence only upon exposure to a specific chemical or environmental stimulus. The promoter may be host specific. The promoter may be suitable for expressing the polynucleotide in plants, bacteria, or yeast. The promoter may be a plant-specific promoter. The promoter may be specific for a particular developmental stage, organ, or tissue. Tissue-specific promoters are capable of initiating transcription in specific plant tissues. Plant tissues that may be targeted by tissue-specific promoters may include, but are not limited to, stems, leaves, trichomes, anthers, or seeds. Constitutive promoters herein may include the rice ubiquitin 3 promoter (OsUbi3P) or the rice actin 1 promoter. Other known constitutive promoters can be used, and include but are not limited to the cauliflower mosaic virus (CAMV) 35S promoter, the Cestrum Yellow Leaf Curling Virus promoter (CMP) or CMP short version (CMPS), the Rubisco small subunit promoter, and the maize ubiquitin promoter. The tissue-specific promoter may include a seed-specific promoter. The seed-specific promoter may be, but is not limited to, the rice GluB4 promoter or the maize zein promoter.

The promoter may be suitable for expressing polynucleotides in bacteria. The promoter may be a T7 RNA polymerase promoter, a LAC promoter, or an arabinose promoter. The promoter may be suitable for expressing polynucleotides in yeast. The promoter may be a GAL promoter or a glucose promoter. Another regulatory element that may be provided is a terminator sequence that terminates transcription. The terminator sequence may be included at the 3' end of the transcription unit of the expression cassette. The terminator may be derived from genes of various plants. The terminator may be the terminator sequence of the nopaline synthase or octopine synthase genes from Agrobacterium tumefaciens. The possible sequence of the terminator may be any other terminator sequence.

The vectors containing the expression cassettes herein may also include additional genetic elements, such as a multiple cloning site to facilitate molecular cloning and a selection marker to facilitate selection.

In one embodiment, the expression cassette can be optimized for expression in plants. The expression cassette can include, consist essentially of, or consist of a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO: 44 (pAG2209), SEQ ID NO: 45 (pAG2210), SEQ ID NO: 46 (pAG2211), SEQ ID NO: 47 (pAG2212), SEQ ID NO: 48 (pAG2216), SEQ ID NO: 49 (pAG2217), SEQ ID NO: 50 (pAG2218), SEQ ID NO: 51 (pAG2219), SEQ ID NO: 52 (pAG2220), SEQ ID NO: 53 (pAG2221), SEQ ID NO: 54 (pAG2222), and SEQ ID NO: 55 (pAG2223). The reference sequences in the group consisting of NO: 55 (pAG2223) have sequences that are at least 70%, 72%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical.

In one embodiment, the expression cassettes herein can be optimized for expression in bacteria. For expression in E. coli, the expression cassettes herein can be optimized. The expression cassette may comprise, consist essentially of, or consist of a polynucleotide comprising a sequence at least 70%, 72%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference sequence selected from the group consisting of SEQ ID NO: 60 (pHT01-pre-proSavinase-8His), SEQ ID NO: 62 (pHT01-proSavinase-8His), SEQ ID NO: 64 (pHT01-Savinase-8His), SEQ ID NO: 66 (pHT43-pre-proSavinase-8His), SEQ ID NO: 68 (pHT43-proSavinase-8His), and SEQ ID NO: 70 (pHT43-Savinase-8His). The polynucleotide may encode a protease comprising an amino acid sequence that is at least 70%, 72%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a reference sequence selected from the group consisting of SEQ ID NO: 61 (pHT01-pre-proSavinase-8His), SEQ ID NO: 63 (pHT01-proSavinase-8His), SEQ ID NO: 65 (pHT01-Savinase-8His), SEQ ID NO: 67 (pHT43-pre-proSavinase8His), SEQ ID NO: 69 (pHT43-proSavinase-8His) and SEQ ID NO: 71 (pHT43-Savinase-8His).

In one embodiment, the expression cassettes herein can be optimized for expression in yeast. The expression cassettes can comprise, consist essentially of, or consist of a polynucleotide comprising a sequence that is at least 70%, 72%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the reference sequence of SEQ ID NO: 629 (pET22_iSAV_Hwa_S317_nuc) or SEQ ID NO: 630 (P416GALL-Ura).

One embodiment includes a vector containing an expression cassette. The vector can be suitable for transforming an appropriate host. The appropriate host can be, but is not limited to, a plant, a bacterium, or a yeast.

In one embodiment, the intein-modified protease can be expressed in any host.

One embodiment includes a host genetically engineered to express any of the intein-modified proteases described herein. The intein-modified protease can include any target protease. The target protease can be selected from the group consisting of: EC 3.4.99 proteases, EC 3.4.21.62 proteases, keratinase, serine proteases, alkaline proteases, metalloproteases, cysteine proteases, aspartic proteases, ATP-dependent proteases, and subtilisin family proteases. The host can express keratinase. The host can express savinase.

The host can be a cell. The cell can be, but is not limited to, a plant cell, a microbial cell, a fungal cell, a mammalian cell, or an insect cell. The host can also be a bacteriophage or a virus.

The host can be a microorganism. The microorganism can be, but is not limited to, Bacillus subtilus, Bacillus lentus, Bacillus licheniformis, Escherichia coli, Saccharomyces ssp., S. cerevisiae, Pichia ssp., or Pichia pastoris.

The host can be a plant. The plant can be, but is not limited to, corn, soybean, sorghum, switchgrass, sugarcane, wheat, alfalfa, barley or rice.

The host can be an expression host. The expression host can be tested using standard methods known in the art. The expression host can be a microbial expression host. The microbial expression host can be a unicellular bacterium. The expression host can be a fungal host or an archaeal host, a plant expression host, an insect cell expression host, a viral expression host, a phage expression host or a mammalian expression host. The intein-modified protease can be expressed in the expression host or expressed in vitro in an expression system. Microbial expression hosts are generally preferred because of their ease of use and the wide range of technological platforms that are easily accessible to these organisms. Microbial expression hosts can include, but are not limited to, Bacillus subtilis, Bacillus lentus, Bacillus licheniformis, Escherichia coli, Saccharomyces cerevisiae, Saccharomyces cerevisiae, Pichia pastoris or Pichia pastoris and other microbial expression hosts well known in the art.

One embodiment includes a method for detecting an expressed intein-modified protease. Detection can include at least one of: analyzing the level of mRNA encoding the intein-modified protease in the expression host by RT-PCR or Northern analysis, analyzing the level of the intein-modified protease in the expression host or in the host growth medium by Western analysis or mass spectrometry, or by measuring the activity level of the pre-spliced or spliced protease in the expression host, host tissue, or host growth medium. Protease activity can be detected by a number of different methods, including methods using labeled substrates or by tracking the concentration of the target protease on a Coomassie-stained gel after electrophoresis.

One embodiment provides a method for preparing a protease. The method includes causing splicing of an intein-modified protease, wherein the intein-modified protease can be any intein-modified protease described herein. The method can include obtaining the intein-modified protease.

The obtaining step may include genetically engineering the host by transformation using an expression cassette encoding an intein-modified protease. The transformation may be, but is not limited to, Agrobacterium-mediated transformation, electroporation using plasmid DNA, DNA uptake, biolistic transformation, virus-mediated transformation, or protoplast transformation. The transformation may be any other transformation method suitable for the particular host.

In one embodiment, the method can include the step of preparing an expression cassette before conversion. The step of preparing the expression cassette can include selecting a target protease. The target protease can be selected from the group consisting of EC 3.4.99 protease, EC 3.4.21.62 protease, keratinase, serine protease, alkaline protease, metalloprotease, cysteine protease, aspartic protease, ATP-dependent protease and subtilisin family protease. The step of preparing the expression cassette can include directly inserting a polynucleotide encoding an intein into the nucleic acid encoding the target protease before one or more portions of the encoding cysteine, serine or threonine residue sequence.

A polynucleotide encoding an intein can be inserted into the portion of a nucleic acid encoding the catalytic domain of a target protease. Insertion of the intein into the catalytic domain can inactivate the target protease. A polynucleotide encoding an intein can be inserted into the portion of a nucleic acid encoding the splitting site of the catalytic domain. The splitting site can include an amino acid site characterized by one or more of the following: 1) on the surface of the secondary structure of the target protease; 2) near the end of the secondary structure; 3) between catalytic residues of the catalytic domain; or 4) near the end of the catalytic domain.

The step of preparing the expression cassette can include modifying the gene encoding the intein-modified protease using recombinant DNA methods, PCR, or by synthesizing a nucleic acid encoding the desired intein-modified protease. Using any of these methods, a nucleic acid sequence encoding the amino acid sequence of the intein can be assembled or fused within a nucleic acid sequence encoding the desired target protease or a portion of the target protease. When using a cis-spliced intein, the resulting intein-modified protease nucleic acid sequence can encode a continuous amino acid sequence, or when using a trans-spliced intein, two separate nucleic acids encoding an intein-extein fusion can be obtained. For any target protease and a selected insertion site, any desired intein, even novel or engineered inteins, can be inserted. Inserting multiple inteins into the selected insertion site of a target protease provides a method for screening and selecting intein-modified proteases with desired activity characteristics. This process for developing intein-modified proteases can be enhanced or improved by using directed mutagenesis, random mutagenesis, or DNA shuffling to create intein-modified protease libraries that can be screened to identify desired intein-modified proteases. This method can be used to select intein-modified proteases that can be expressed in an inactive state at 37°C when formulated in detergent and become active again at temperatures below 34°C or 20°C when the detergent is diluted in water.

In one embodiment, the intein can spontaneously splice the intein-modified protein. The intein can cause trans-splicing of the intein-modified protease when the first portion and the second portion of the intein-modified protease contact each other.

In one embodiment, upon exposure to inducing conditions, the intein can be induced to cause cis-splicing of the intein-modified protease.

The step of causing may comprise allowing the intein to be spontaneously spliced to splice the intein-modified protease. The step of causing may comprise inducing splicing by exposing the intein-modified protease to inducing conditions.

The induction conditions may be selected from the group consisting of an induction temperature, an induction pH, an induction concentration of a compound, an induction compound, and an induction mixture of compounds. The induction conditions may be an induction temperature. The induction temperature for the intein may be a temperature below 37°C. The induction temperature may be a temperature below 28°C, below 25°C, or below 20°C. The induction temperature may be a temperature below or equal to 20°C. The induction temperature may be 37°C, 35°C, 30°C, 25°C, 20°C, less than 37°C, less than 35°C, less than 30°C, less than 25°C, less than 20°C, 37°C to 35°C, 35°C to 30°C, 30°C to 25°C, 25°C to 20°C, or a temperature less than 20°C.

The inducing condition can be an inducing concentration of the compound. The inducing concentration of the compound can be a decrease in concentration. The decrease can be, but is not limited to, a decrease of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%. The inducing concentration of the compound can be an increase in concentration. The increase can be, but is not limited to, an increase of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100% or more. The inducing compound can be selected from the group consisting of detergents, surfactants, chelating agents, zinc, EDTA, ions, and phytic acid.

The inducing compound can be a detergent. As used herein, "detergent" refers to a surfactant or a mixture of surfactants. The surfactant can be an alkylbenzenesulfonate. The detergent can be a laundry detergent. The laundry detergent can contain water softeners, surfactants, bleaching agents, enzymes, whitening agents, spices, and many other agents. Laundry detergents can be provided in the form of liquid formulations. Liquid laundry detergent formulations can include anionic and nonionic surfactants, builders to remove hardness ions, various anti-redeposition agents, dye transfer inhibitors to prevent dyes from falling off one fabric and depositing on another, soil release polymers to provide a barrier for the fabric, fluorescent brighteners, enzyme stabilizers, viscosity control compounds, pH control compounds, soaps and polysiloxanes to control excessive foaming, preservatives to control microorganisms, perfumes and dyes to impart odor and appearance, bleaching agents, water, solubilizers, and other additives to improve performance characteristics. The anionic surfactant can be an alkylbenzenesulfonate. The nonionic surfactant can be an ethoxylated fatty alcohol or any other anionic surfactant. The hardness ion removal builder can be sodium citrate, tetrasodium EDTA, or an acrylic polymer. The dye transfer inhibitor can be PVP K-30, Chromabond S-100, or Chromabond S-400. The soil release polymer can be Sorez 100 (a copolyester of polyethylene glycol) or Repel-O-Tex SRP-6 (a polyethylene glycol polyester). The fluorescent whitening agent can be Tinopal CBS-X or any other fluorescent whitening agent. The enzyme stabilizer can be calcium chloride, sodium tetraborate, propylene glycol, sodium formate, sodium citrate, or monoethanolamine. The viscosity control compound can be propylene glycol, sodium xylene sulfonate, or a polymer. The pH control compound can be citric acid or monoethanolamine. The detergent can be affected by the temperature of the wash water. The detergent can be a dishwashing detergent. The detergent can also be a soapless soap. The term "soapless soap" refers to a soapless liquid detergent with a slightly acidic pH. The detergent can be a cleaning solution. The cleaning solution can be an industrial cleaning solution or a commercial cleaning solution. The cleaning solution can be a cleaning solution for cleaning fabrics, clothing, textiles, dishes, cutlery, consumer or industrial products, pipes, equipment, scaling, or biofilm. The detergent can be any other type of detergent. The detergent can also be a powder. The detergent can be a liquid solution. The detergent can be a fuel additive. The fuel additive can be a long-chain amine or amide. The fuel additive can be polyisobuteneamine or polyisobuteneamide/succinimide. The detergent can be a biological reagent. The biological reagent can be used for the separation and purification of integral membrane proteins found in biological cells.

The intein-modified protease is inactivated when diluted with detergent and is activated upon dilution of the detergent with water.

In one embodiment, the step of causing can include achieving splicing of the intein under conditions where a detergent is diluted with water. The ratio of detergent to water can be selected from less than or equal to any one of the following values: 1:5, 1:10, 1:20, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, or 1:400, or any value in a range between any two of the foregoing values (including endpoints). For example, the ratio of detergent to water can be less than any integer or non-integer value selected from between 1:5 and 1:10. The ratio of the detergent to water can be 1:5, 1:10, 1:20, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350 or 1:400, or any value in the range between any of the foregoing (including endpoints). For example, the liquid-to-solid ratio can be a value equal to any integer or non-integer selected from between 1:5 and 1:10.

One embodiment includes a method for regulating the activity of a protease, including preparing the protease by any of the methods described herein. The protease activity can be regulated during expression, purification, formulation, or in the final product. The protease activity can be regulated during industrial, consumer, agricultural, or animal husbandry processes. The consumer process can be the process of cleaning consumer products. The consumer process can be the washing of clothing or fabric items, washing dishes, or other materials. The agricultural or animal husbandry process can include the preparation of meat, protein, eggs, milk, other dairy products, poultry, pig, or cattle products. The use of controllable proteases can increase the value of the animal husbandry process. The use of inteins to modify proteases and thereby regulate when and how they are activated is a new means for increasing the production and use of proteases and discovering new proteases. Depending on the desired application, both cis- and trans-spliced inteins are valuable when preparing, formulating, and using with proteases. The cis-spliced intein can be fused to the target protease, it can be located internally (i.e., inserted into the amino acid sequence of the protease), or it can be fused to the amino terminus or carboxyl terminus of the target protease. The intein is inserted into or fused to the target protease so that the activity of the protease can be regulated by intein cleavage, splicing, or even by changing the conformation of the intein-modified protease. The activation of the intein-mediated protease can be regulated by inducing conditions. The inducing conditions can include conditions sufficient to induce splicing of the intein within the intein-modified protease. The activation of the intein-mediated protease can be regulated by the protease expression host. In one embodiment, the activation of the intein-mediated protease may occur spontaneously.

Intein-modified proteases capable of cis-splicing can be expressed and formulated into detergents at elevated temperatures, where splicing is inhibited or the protease is inactivated. Upon dilution of the detergent containing the intein-modified protease, the splicing or cleavage reaction can proceed, activating the protease.

Intein-modified proteases are capable of trans-splicing. Toxic compounds are often manipulated as binary systems, separated into two inactive parts, prepared and stored separately, and brought together to form a functional compound when needed. Proteases can be separated into two inactive parts and reassembled into functional molecules using trans-splicing inteins. See Kempe et al. (2009), which demonstrates the use of trans-splicing inteins for the expression of a cytotoxic barnase from Bacillus amyloliquefaciens in plants.

Intein-modified proteases can be expressed as inactive precursors but may subsequently be activated during the formulation of a diet for animal feeding, or to form active proteases in the animal body that improve the nutritional properties of the feed. Other mechanisms for activating intein-modified proteases are also possible, depending on the desired conditions and application.

In one embodiment, a trans-splicing intein can be inserted into a protease, separating it into two intein-modified extein proteins. Upon binding, the two intein-modified extein proteins can combine to activate the protease, or splice via intein cis-splicing to produce a mature, active enzyme. Intein trans-splicing enables methods for preparing proteases in which an active protease can be assembled in vitro or in vivo from two inactive, or barely active, precursor intein-modified exteins. By using trans-splicing, it is possible to select intein-modified protease exteins that can bind in a dry form, thus being inactive, and then become activated upon hydration. This formulation mechanism is useful in animal feed and dry detergent formulations. Similarly, intein-modified protease exteins can be formed in high concentrations of detergents or other chemicals, where the protease may not be activated in the initial formulation but becomes active upon dilution. Other chemicals may include zinc, EDTA, anions, cations, chelating agents, fatty acids, phytic acid, surfactants, or other chemicals known in the art.

One embodiment includes a detergent containing an intein-modified protease. The intein-modified protease can include any target protease herein. The target protease can be Savinase.

One embodiment includes an animal feed containing an intein-modified protease, which can be any of the intein-modified proteases described herein. For use as an animal feed or as a portion of an animal feed, the intein-modified protease can be produced by a microorganism. The intein-modified protease can be produced in plants. The target protease can be, but is not limited to, an enzyme used as a dietary supplement or a mixture of exogenously produced enzymes. Expressing the intein-modified protease in soybean, corn, rye, wheat, or sorghum seeds can eliminate the need for exogenously produced enzymes that must be incorporated into the feed and is a more efficient way to prepare animal feed containing grains containing the target protease. Expressing the intein-modified protease in seeds or grains can facilitate the incorporation of enzymes or enzyme mixtures into dietary supplements for nutritional enhancement. Enzymes and enzyme mixtures have been increasingly used as additives in animal feed to increase nutrient availability, eliminate some anti-nutritional effects in feed, and modulate the intestinal flora, particularly in young animals, which are most susceptible to enteric pathogens (Bedford and Partridge, 2010). The intein-modified protease can be produced in seeds, and the seeds or intein-modified protease can be at least part of an animal feed.

The intein-modified proteases can be mixed with other enzymes reported as dietary additives in pig and poultry feeds. The intein-modified proteases can be mixed with any one or more of xylanases, β-glucanases, proteases, amylases, phytases, or endo-mannanases (Cowieson et al., 2005; Mathlouthi et al., 2002; Jiang et al., 2008; Liu et al., 2008a, b; Short F.M. et al., 2002; Odetallah et al., 2002a, b; Wang et al., 2006a; and Stark et al., 2009). The intein-modified proteases can be mixed with other feed additives. The intein-modified proteases can be mixed with other proteases included in various animal feed diets. The intein-modified proteases can be used alone or as a mixture of enzymes in various poultry feed diets. The beneficial effects of protease supplementation have been documented in terms of improving feed utilization, growth performance, and reducing mortality in immature and growing animals. See Simbaya et al., 1996, Odetallah et al., 2003, and Wang et al., 2006, all of which are incorporated by reference as if fully set forth. Intein-modified proteases can be mixed with other commercially available protease-fortified poultry feeds, including but not limited to various feeds (Danisco) or keratinase PWD-1 (BioResource International, Inc.). Protease-fortified poultry feeds can be used as target proteases.

One embodiment includes an intein-modified protease comprising keratinase as the target protease. Keratinases appear to have independently and centrally evolved Asp/Ser/His catalytic triads, similar to the catalytic triad found in the trypsin serine protease (PFAM 00089). PWD-1 keratinase was isolated and characterized from the feather-degrading bacterium Bacillus licheniformis PWD-1 (Lin et al., 1992). Bacillus licheniformis PWD-1 keratinase (Q53521) belongs to the subtilisin family of serine proteases. This enzyme has been used to produce hydrolyzed feather meal and has potential applications in various applications in the animal feed, leather, fertilizer, detergent, and pharmaceutical industries (Gupta and Ramnani, 2006; Brandelli, 2008, Brandelli et al., 2010). For use as a feed additive, keratinase is prepared as a natural, dried, cell-free fermentation product derived from the keratinase-producing Bacillus licheniformis PWD-1. It would be beneficial to produce keratinase directly in forage plants, crops, tissues, or cereals, rather than using microbially produced keratinase as a feed additive. Keratinase can be produced in any transgenic forage plant, crop, tissue, or cereal, including corn (stalks and/or cereals), sorghum (cereals, forage, and/or residues), or soybeans. However, keratinase is difficult to produce inherently due to potential hazards to the expression host. Expressing active keratinase can be harmful to plant cells, and transgenic events expressing high levels of keratinase are inhibited from selection and fail. This can be seen as agenesis or defective seed development. To date, there are no reports of plant-expressed keratinase. Among the challenges in producing functional keratinase is the cleavage of the precursor protein and the proteolytic degradation of the inhibitory pro-domain. When cleavage is rate-limiting, the cleavage site of known proteases activated in the plant secretory pathway can be located between the pro-domain and the catalytic domain. Conversely, the pro-enzyme can be targeted to the vacuole, where non-secretory pathway proteases are sequestered. Alternatively, auto-processing can be induced by inactivating small amounts of activated keratinase in the material at the post-harvest stage.

One embodiment includes a keratinase modified for expression in plants. The modified keratinase can be an intein-modified keratinase. The intein-modified keratinase can contain a trans-spliced intein.

One embodiment includes a method for preparing a keratinase that can be expressed in plants. Because keratinase can have an adverse effect on seed development, keratinase can be divided into inactive supplementary parts and reassembled to obtain activity using trans-splicing inteins when used. Keratinase can also be expressed as a cis-splicing intein-modified protease. Keratinase can be modified to express a prodomain and a catalytic domain separately. In the absence of the prodomain, the catalytic domain can be modified to fold incorrectly. The two domains can then be merged. Keratinase belongs to the subtilisin family, which includes proteases that are self-processed through a complex maturation pathway. Subtilisin is synthesized as an inactive pro-precursor protein with an N-terminal signal peptide followed by the prodomain and catalytic domain (Takagi and Takahashi, 2003). The signal peptide is removed during the secretion process that produces the inactive proprotein across the cytoplasmic membrane. The prodomain is also removed by autocatalysis or by an active subtilisin molecule to produce a functional protein (Ohta et al., 1991; Carter and Wells, 1988). The rate-limiting step in maturation is not the folding or self-processing of the proprotein to produce the catalytic protease, but rather the release of the first enzymatically active catalytic protease from the inhibitory prodomain associated with it by degradation of the prodomain (Yabuta et al., 2001). This triggers a chain reaction that selectively and exponentially degrades the prodomain and enhances activity. This shows that the prodomain can play a trans role in the catalytic domain of the folded inactive molten globule to activate the conformation. See Baker et al., 1992; and Shinde and Inouye, 1995. It is useful to control activity by expressing the prodomain and catalytic domain separately. Keratinase can be divided into two parts: the prodomain and the catalytic domain. The catalytic domain can also be split into two parts to inactivate the keratinase. The cleavage site can include an amino acid site characterized by being on the surface of the secondary structure of the target protease, near the end of the secondary structure between the catalytic residues of the catalytic domain, or near the end of the catalytic domain. Table 1 describes the Q53521 keratinase sites in plants for potential secondary modification. These sites can be altered by conservative substitutions of amino acid residues, or by other means that reduce the probability of post-translational modification of the plant-expressed protease.

Table 1. Potential secondary modification sites of Q535211

Prediction tools are available at http://expasy.org/proteomics: NetNGlyc1.0, NetOglyc3.1, NetPhos2.0, NetPhosK1.0, OGPET, YinOYang1.2, Big-PI, NMT, Pre-PS, and Sulfinator were applied to the full-length Q53521. Matches are listed in Table 1.

The keratinase can be divided into an N-extein and a C-extein. Each N-extein and C-extein can be fused to a trans-spliced intein portion to form an inactive cis-spliced NI pair and IC pair. The NI pair and IC pair can be expressed under the control of an inducible promoter in male and female plants during the early germination stage. Hybridization of the plants can produce hybrid seeds that do not express the keratinase and develop normally. Seed imbibition can induce co-expression of the inactive, reassembled trans-spliced portions, and active keratinase can be generated early in germination. Separately expressing the trans-spliced portions of intein-modified keratinase has the following advantages: i) expression in germinating seeds does not affect normal seed development and seed set; ii) the keratinase can promote protein degradation in seeds and potentially improve the nutritional value of feed; iii) the accompanying sugar mobilization from starch in germinating seeds can further enhance nutritional value; iv) the production of keratinase using hybrid seed production techniques offers increased value; v) the toxicity of keratinase in germinating seeds can be advantageous, providing a means to control the spread of functional genes; and vi) for plant expression, it is possible to modify the gastric-labile cis-spliced Q53521 portions (NI and IC) and regain sufficient stability of the active keratinase for protein degradation in the animal gut. It is also possible to express the keratinase directly in green plant tissues, where it is targeted to the cell wall and does not interfere with other cellular or plant functions. The expression of trans-spliced portions of proteases in male and female plants, respectively, under the control of a germination-inducible promoter, has broad utility for the expression of deleterious proteases in seeds that affect normal seed development and/or fertility and fruit set.

Embodiments include compositions produced by one or more steps of the methods described herein.

Implementation Method

The following items include specific embodiments. However, they are not intended to limit or exclude the embodiments described elsewhere herein or alternative embodiments.

1. An intein-modified protease, comprising a target protease and an intein fused to the target protease, wherein the fusion position enables the intein to control the activity of the target protease, wherein the intein is capable of achieving splicing of the intein-modified protease.

2. The intein-modified protease according to embodiment 1, wherein the position of the fusion enables the intein to substantially reduce or inhibit the activity of the target protease.

3. The intein-modified protease according to any one or more of embodiments 1-2, wherein the target protease is an enzyme selected from the group consisting of: EC 3.4.99 protease, EC 3.4.21.62 protease, keratinase, serine protease, alkaline protease, metalloprotease, cysteine protease, aspartic acid protease, ATP-dependent protease and subtilisin family protease.

4. The intein-modified protease according to any one or more of embodiments 1-3, wherein the target protease comprises a keratinase.

5. The intein-modified protease according to any one or more of embodiments 1-3, wherein the target protease comprises Savinase.

6. The intein-modified protease of any one or more of embodiments 1-5, wherein the target protease comprises a sequence that is at least 90% identical to a reference sequence selected from the group consisting of SEQ ID NOs: 1-12, 57-58, and 718.

7. The intein-modified protease of any one or more of embodiments 1-6, wherein the intein is capable of trans-splicing the intein-modified protease, and the intein-modified protease comprises: i) a first portion having an N-extein of a target protease and an N-intein of an intein, with the carboxyl terminus of the N-extein fused to the amino terminus of the N-intein; and ii) a second portion having a C-intein of the intein and a C-extein of the target protease, with the carboxyl terminus of the C-intein fused to the amino terminus of the C-extein; wherein the first portion and the second portion are separated prior to splicing of the intein-modified protease.

8. The intein-modified protease of any one or more of embodiments 1-7, wherein the N-intein comprises a sequence that is at least 90% identical to a reference sequence selected from the group consisting of SEQ ID NO: 38, SEQ ID NO: 537, SEQ ID NO: 539, SEQ ID NO: 541, and SEQ ID NO: 543.

9. The intein-modified protease of any one or more of embodiments 1-8, wherein the C-intein comprises a sequence that is at least 90% identical to a reference sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 538, SEQ ID NO: 540, SEQ ID NO: 542, and SEQ ID NO: 544.

10. The intein-modified protease of any one or more of embodiments 1-8, wherein the first portion comprises a peptide sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 454, SEQ ID NO: 456, SEQ ID NO: 457, SEQ ID NO: 458, SEQ ID NO: 459, SEQ ID NO: 460, SEQ ID NO: 461, SEQ ID NO: 462, SEQ ID NO: 463, SEQ ID NO: 464, SEQ ID NO: 465, SEQ ID NO: 466, SEQ ID NO: 467, SEQ ID NO: 468, SEQ ID NO: 469, SEQ ID NO: 470, SEQ ID NO: 471, SEQ ID NO: 472, SEQ ID NO: 473, SEQ ID NO: 474, and SEQ ID NO: 475. The reference sequences in the group consisting of NO: 475 are sequences with at least 90% identity.

11. The intein-modified protease of any one or more of embodiments 1-11, wherein the second portion comprises a peptide sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 455, SEQ ID NO: 476, SEQ ID NO: 477, SEQ ID NO: 478, SEQ ID NO: 479, SEQ ID NO: 480, SEQ ID NO: 481, SEQ ID NO: 482, SEQ ID NO: 483, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486, SEQ ID NO: 487, SEQ ID NO: 488, SEQ ID NO: 489, SEQ ID NO: 490, SEQ ID NO: 491, SEQ ID NO: 492, SEQ ID NO: 493, SEQ ID NO: 494, and SEQ ID NO: 495. The reference sequences in the group consisting of ID NO: 495 have sequences with at least 90% identity.

12. The intein-modified protease according to any one or more of embodiments 1-6, wherein the intein is capable of achieving cis-splicing of the intein-modified protease.

13. The intein-modified protease of any one or more of embodiments 1-6 and 12, wherein the intein comprises a sequence that is at least 90% identical to a reference sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 119, SEQ ID NOs: 497-533, and SEQ ID NOs: 684-685.

14. The intein-modified protease according to any one or more of embodiments 1-6, 12 and 13, comprising a sequence that is at least 90% identical to a reference sequence selected from the group consisting of SEQ ID NOs: 25-36, SEQ ID NOs: 120-453, SEQ ID NO: 496 and SEQ ID NOs: 686-696.

15. The intein-modified protease of any one or more of embodiments 1-6 and 12-14, wherein the intein can inducibly cause cis-splicing of the intein-modified protease by exposure to inducing conditions, wherein the inducing conditions are selected from at least one of the group consisting of: inducing temperature, inducing pH, inducing concentration of a compound, an inducing compound, and an inducing mixture of compounds.

16. The intein-modified protease according to any one or more of embodiments 1-6 and 12-15, wherein the induction condition is an induction temperature, and the induction temperature is a temperature below 37°C.

17. The intein-modified protease according to any one or more of embodiments 1-6 and 12-16, wherein the induction temperature is a temperature below 28°C, below 25°C or below 20°C.

18. The intein-modified protease of any one or more of embodiments 1-6 and 12-15, wherein the inducing condition is an inducing concentration of a compound, wherein the compound is selected from the group consisting of: detergents, surfactants, chelating agents, zinc, EDTA, and phytic acid.

19. The intein-modified protease of any one or more of embodiments 1-6, 12-15, and 18, wherein the intein can inducibly cause cis-splicing of the intein-modified protease by exposure to inducing conditions, wherein the inducing conditions include an inducing compound and an inducing concentration of the compound, and wherein the inducing compound is a detergent, and the inducing concentration of the detergent diluted with water is a detergent:water ratio less than or equal to one selected from the group consisting of: 1:5, 1:10, 1:20, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, and 1:400.

20. An expression cassette comprising a polynucleotide encoding an intein-modified protease, wherein the intein-modified protease comprises a target protease and an intein fused to the target protease, wherein the fusion position enables the intein to control the activity of the target protease, wherein the intein is capable of achieving splicing of the intein-modified protease.

21. The expression cassette of embodiment 20, wherein the position of the fusion is such that the intein is capable of substantially reducing or inhibiting the activity of the target protease.

22. The expression cassette of any one or more of embodiments 20-21, wherein the polynucleotide comprises a sequence encoding a keratinase.

23. The expression cassette of any one or more of embodiments 20-21, wherein the polynucleotide comprises a sequence encoding Savinase.

24. The expression cassette of any one or more of embodiments 20-23, wherein the polynucleotide comprises a sequence that is at least 90% identical to the reference sequence of SEQ ID NO: 41 or SEQ ID NO: 59.

25. The expression cassette of any one or more of embodiments 20-24, wherein the polynucleotide comprises a sequence encoding an intein capable of trans-splicing an intein-modified protease, and the intein-modified protease comprises: i) a first portion having an N-extein of a target protease and an N-intein of an intein, with the carboxyl terminus of the N-extein fused to the amino terminus of the N-intein; and ii) a second portion having a C-intein of the intein and a C-extein of the target protease, with the carboxyl terminus of the C-intein fused to the amino terminus of the C-extein; wherein the first portion and the second portion are separated prior to splicing of the intein-modified protease.

26. The expression cassette of any one or more of embodiments 20-25, wherein the sequence is at least 90% identical to a reference sequence selected from the group consisting of SEQ ID NOs: 42-43 and SEQ ID NOs: 674-681.

27. The expression cassette of any one or more of embodiments 20-26, wherein the sequence is at least 90% identical to a reference sequence selected from the group consisting of SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 587, and SEQ ID NOs: 569-608.

28. The expression cassette of any one or more of embodiments 20-27, wherein the polynucleotide comprises a sequence that is at least 90% identical to a reference sequence selected from the group consisting of SEQ ID NO: 94, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 588, and SEQ ID NOs: 609-628.

29. The expression cassette of any one or more of embodiments 20-24, wherein the polynucleotide comprises a sequence encoding an intein capable of cis-splicing a protease that modifies the intein.

30. The expression cassette of any one or more of embodiments 20-24 and 29, wherein the polynucleotide comprises a sequence that is at least 90% identical to a reference sequence selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 72, SEQ ID NOs: 105-106, SEQ ID NO: 119, SEQ ID NOs: 634-673, and SEQ ID NOs: 699-700.

31. The expression cassette of any one or more of embodiments 20-24, 29, and 30, wherein the polynucleotide comprises a sequence that is at least 90% identical to a reference sequence selected from the group consisting of SEQ ID NOs: 73-92, SEQ ID NOs: 107-118, SEQ ID NOs: 545-586, and SEQ ID NOs: 701-711.

32. The expression cassette of embodiment 20, wherein the intein-modified protein is the intein-modified protein of any one of embodiments 2-19.

33. An expression cassette comprising a polynucleotide comprising a sequence having at least 90% identity to a reference sequence selected from the group consisting of SEQ ID NOs: 44-55.

34. An expression cassette comprising a polynucleotide comprising a sequence at least 90% identical to the reference sequence of SEQ ID NO: 629 or SEQ ID NO: 630.

35. A host genetically engineered to express the intein-modified protease of any one of embodiments 1-19.

36. The host of embodiment 35, wherein the host is selected from the group consisting of a plant cell, a microbial cell, a fungal cell, a mammalian cell, a bacteriophage, a virus, and an insect cell.

37. The host of any one or more of embodiments 35-36, wherein the host is a microorganism selected from the group consisting of: Bacillus subtilus, Bacillus lentus, Bacillus licheniformis, Escherichia coli, Saccharomyces sp., Saccharomyces cerevisiae, Pichia ssp., and Pichia pastoris.

38. The host of any one or more of embodiments 35-36, wherein the host is a plant selected from the group consisting of corn, soybean, sorghum, switchgrass, sugarcane, wheat, alfalfa, barley, and rice.

39. A method for preparing a protease, the method comprising causing splicing of an intein-modified protease, wherein the intein-modified protease is the intein-modified protease described in any one of embodiments 1-19.

40. The method according to embodiment 39 further includes obtaining the protease modified with the intein.

41. The method of any one or more of embodiments 39-40, wherein the obtaining step comprises genetically engineering the host by transforming the host with an expression cassette encoding an intein-modified protease.

42. The method according to any one or more of embodiments 39-41, further comprising preparing an expression cassette prior to the transforming step.

43. A method according to any one or more of embodiments 39-42, wherein the step of preparing the expression cassette comprises selecting a target protease from the group consisting of: EC3.4.99 protease, EC3.4.21.62 protease, keratinase, serine protease, alkaline protease, metalloprotease, cysteine protease, aspartic acid protease, ATP-dependent protease and protease of the subtilisin family.

44. A method according to any one or more of embodiments 39-43, wherein the step of preparing the expression cassette comprises inserting a polynucleotide encoding an intein directly into the nucleic acid encoding the target protease before one or more portions of the sequence encoding a cysteine, serine or threonine residue.

45. A method according to any one or more of embodiments 39-44, wherein the polynucleotide encoding the intein is inserted into the portion of the nucleic acid sequence encoding the catalytic domain of the target protease, and the insertion of the intein into the catalytic domain inactivates the target protease.

46. A method according to any one or more of embodiments 39-45, wherein the polynucleotide encoding the intein is inserted into the nucleic acid portion encoding the cleavage site of the catalytic domain, wherein the cleavage site includes an amino acid site characterized by one or more of the following: 1) on the surface of the secondary structure of the target protease; 2) near the end of the secondary structure; 3) between catalytic residues of the catalytic domain, or 4) near the end of the catalytic domain.

47. The method of any one or more of embodiments 39-46, wherein the intein is capable of achieving trans-splicing of the intein-modified protease.

48. The method of any one or more of embodiments 39-47, wherein the intein is spontaneously spliced, causing the intein to be spliced, including causing the intein to be spliced.

49. A method according to any one or more of embodiments 38-46, wherein the intein can inducibly cause cis-splicing by exposure to inducing conditions, wherein the inducing conditions include at least one selected from the group consisting of an inducing temperature, an inducing pH, an inducing concentration of a compound, an inducing compound or an inducing mixture of compounds, and the causing includes exposing the intein-modified protein to inducing conditions.

50. The method of any one or more of embodiments 39-46 and 49, wherein the induction condition is an induction temperature, wherein the induction temperature is a temperature below 37°C.

51. The method of any one or more of embodiments 39-46, 49, and 50, wherein the induction temperature is a temperature below 28°C, below 25°C, or below 20°C.

52. The method of any one or more of embodiments 39-46 and 49, wherein the inducing condition is an inducing concentration of a compound, wherein the compound is selected from the group consisting of: detergents, surfactants, chelating agents, zinc, EDTA, and phytic acid.

53. The method of any one or more of embodiments 39-46, 49, and 52, wherein the compound is a detergent and the induced concentration of the detergent diluted with water is a detergent:water ratio less than or equal to one selected from the group consisting of 1:5, 1:10, 1:20, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, and 1:400 detergent to water.

54. The method of any one or more of embodiments 39-53, wherein the activity of the target protease is substantially reduced or inhibited.

55. The method of any one or more of embodiments 39-54, wherein the target protease is restored to activity by splicing of the intein-modified protease.

56. An animal feed comprising the intein-modified protease of any one of embodiments 1-19.

57. A detergent comprising the intein-modified protease according to any one of embodiments 1-19.

Other embodiments herein may be formed by supplementing an embodiment with one or more elements from any one or more other embodiments herein, and/or by replacing one or more elements from one embodiment with one or more elements from one or more other embodiments herein.

Example

The following non-limiting examples are used to illustrate specific embodiments. Each embodiment can be supplemented with one or more details in one or more of the following examples, and/or one or more elements in an embodiment can be replaced with one or more details in the following examples.

Example 1 Experimental Overview

To express a keratinase for animal feed, the following steps were performed: Bacillus licheniformis PWD-1 keratinase (Q53521) was expressed as a pro-enzyme in the endosperm or embryo of maize seeds. The expression cassette was optimized, and different codon-optimized versions of the coding sequence were tested. The promoter, 5'-UTR intron, and targeting signal were evaluated. Fertility, seed set, and seed viability were tested. Multiple T1 seeds from a single event were screened to identify high-expressing enzymes. Molecular mass, cumulative levels, and activity were measured, and the specific activity, MW, pH, and optimal temperature of the seed-expressed keratinase were compared with those of a microbially produced enzyme. The seed-expressed keratinase can be evaluated as an alternative to microbial keratinase in feeding experiments. Inoculation of the keratinase into elite inbred germplasm can serve as the starting point for a commercially viable feed product. As an alternative strategy, the production of the keratinase using cis- or trans-splicing inteins was explored. The separated prodomain and catalytic domain are expressed separately and then mixed to combine the prodomain and catalytic domain, thereby promoting protein refolding and activity recovery.

Example 2 Expression of Q53521 Keratinase Precursor in Corn Seeds

A maize codon-optimized gene for the Q53521 keratinase from Bacillus licheniformis (gi998767) (SEQ ID NO: 12) was synthesized. The codon-optimized Q53521 gene was cloned into pUC57 to construct pUC57:FPROTQ53. To facilitate cloning between the EcoRI and XhoI sites of pBluescript and lambda phage UniZAP XR (Agilent), the XhoI site at bases 898-903 (CTCGAG) was removed by silently mutating C900 to G (on the labeled sequence) using site-directed mutagenesis. The plant expression construct contains an intact XhoI site.

Nucleotide and protein sequences of the maize codon-optimized Q53521 keratinase from Bacillus licheniformis:

The amino acid sequence and domain structure of Q53521 keratinase from Bacillus licheniformis PWD-1 (http://www.uniprot.org) shown below includes an amino-terminal signal peptide between amino acid residues 1-29 (underlined), a protease inhibitory domain or prodomain located between amino acid residues 30-105 (italics), and a catalytic domain or protease domain located between amino acid residues 106-379 (bold).

MMRKKSFWLGMLTAFMLVFTMAFSDSASA (SEQ ID NO: 12)

To express the precursor enzyme, the initial 29 amino acid residue signal peptide was removed and the first methionine was added to the N-terminus to generate pro-Q53521. Codon-optimized pro-Q53521 genes with and without an endoplasmic reticulum retention signal (SEKDEL) were cloned under the control of the strong endosperm-specific promoter rice glutelin B-4 or the maize 27 kDa γ-zein (Z27) promoter. To enhance expression, introns containing 5'UTR sequences were tested from the maize sucrose synthase gene (SS1), maize alcohol dehydrogenase gene (Adh1), and maize ubiquitin 1 gene (UBI1). A total of 12 expression cassettes were prepared (Table 2), including four rice glutelin B-4 promoters: pAG2209 (SEQ ID NO: 44), pAG2210 (SEQ ID NO: 45), pAG2211 (SEQ ID NO: 46), and PAG 2212 (SEQ ID NO: 47), and eight zein Z27 promoters: pAG2216 (SEQ ID NO: 48), PAG 2217 (SEQ ID NO: 49), pAG2218 (SEQ ID NO: 50), pAG2219 (SEQ ID NO: 51), pAG2220 (SEQ ID NO: 52), pAG2221 (SEQ ID NO: 53), pAG2222 (SEQ ID NO: 54), and pAG2223 (SEQ ID NO: 55). The expression cassette was cloned into the KpnI-AvrII site of the basic transformation vector pAG2005 (SEQ ID NO: 56), which carries a spectinomycin resistance marker, a bacterial origin of replication, and the right (RB) and left (LB) Agrobacterium T-DNA borders. Between the RB and LB is a multiple cloning site (MCS) and a plant selectable marker comprising a rice Ubi3 promoter (OsUbi3P), a phosphomannose isomerase coding sequence, and a Nos terminator. This plasmid also carries the MCS, supplemented by the rice Ubi3 promoter (OsUbi3P) and Nos terminator, between which additional coding sequences can be added.

Table 2. List of plant expression cassettes and genetic elements of expression cassettes

As previously mentioned (Komari et al., 2006), Agrobacterium tumefaciens super binary vector (superbinary vector) is used for plant transformation. The expression cassette is cloned between the T-DNA borders of the pSB11 vector (Japan tobacco) with the plant selective PMI expression cassette (Privalle, 2002). Before transformation, each expression cassette is sequence verified. The pSB11 vector carrying the plant expression cassette in the T-DNA border is imported into the Agrobacterium tumefaciens LBA4404 that retains PSB1, to generate the super binary vector of the sheath integration for transformation. The transformation of maize immature embryos is carried out according to the Japan tobacco protocol (Ishida et al., 1996,2007).

Five expression cassettes were introduced into pAG2209, pAG2210, pAG2212, pAG2216, and pAG2217 vectors, respectively, and transformed into maize to generate transgenic events. Events were verified by genotyping for the internal fragment of the PMI selectable marker and pro-Q53521.

The T0 plant shows normal growth and development and normal reproduction. Transgenic plants (T0) are backcrossed with AxB wild-type corn plants and harvested in the crops T1 seeds. Variable fruiting and seed weight were observed in the progeny, but no signs of change were found to be caused by transgenic expression. Transgenic events can be used as pollen donors (male) or pollen receptors (female). The hybridization used as pollen receptors (female) in the transgenic events generally performs better. Table 3 shows the vector events with the quantity of T1 seeds and the total seed weight. In this table, a column with the title "vector event" has included the numbering of the vector used to produce this event, as well as the numbering of the transgenic event assigned. For example, the number 2209_6 in the table represents the transgenic event created using the pAG2209 vector, and the event so obtained is numbered 6.

Table 3. Top five Q53521 transgenic events per vector ranked by T1 seed yield.

Carrier_Event Expression cassette Seed weight (g) Number of seeds Hybrid type 2209_6 GlutB:GluB-4:Q53521 59.60 298 male 2209_7 GlutB:GluB-4:Q53521 55.60 287 male 2212_19 GlutB:GluB-4:Q53521:SEKDEL 52.20 361 male 2212_14 GlutB:GluB-4:Q53521:SEKDEL 49.70 302 male 2209_2 GlutB:GluB-4:Q53521 49.30 277 male 2210_11 GlutB:GluB-4:Q53521:SEKDEL 48.10 229 male 2212_15 GlutB:GluB-4:Q53521:SEKDEL 45.00 297 male 2209_9 GlutB:GluB-4:Q53521 44.90 411 male 2209_11 GlutB:GluB-4:Q53521 43.90 387 male 2212_4 GlutB:GluB-4:Q53521:SEKDEL 43.20 228 male 2210_3 GlutB:GluB-4:Q53521:SEKDEL 42.70 261 male

2212_23 GlutB:GluB-4:Q53521:SEKDEL 42.60 321 male 2210_5 GlutB:GluB-4:Q53521:SEKDEL 38.70 289 female 2217_107 mZein:AdhI:mZ27:Q53521 38.40 285 male 2210_5 GlutB:GluB-4:Q53521:SEKDEL 36.70 328 male 2217_110 mZein:AdhI:mZ27:Q53521 32.30 203 male 2210_17 GlutB:GluB-4:Q53521:SEKDEL 29.70 287 male 2217_103 mZein:AdhI:mZ27:Q53521 27.60 168 male 2216_5 mZein:mSSI:mZ27:Q53521 24.50 210 male 2217_108 mZein:AdhI:mZ27:Q53521 19.30 142 male 2217_104 mZein:AdhI:mZ27:Q53521 17.90 216 male 2216_1 mZein:mSSI:mZ27:Q53521 10.10 150 male 2216_4 mZein:mSSI:mZ27:Q53521 5.30 37 female 2216_5 mZein:mSSI:mZ27:Q53521 0.10 1 female

A sampling procedure was established that allowed genotyping and testing for keratinase activity in the dry seed endosperm while maintaining seed viability. Seeds were split in half for sampling. The embryo-containing portion of the seed was retained and the other portion was tested. The embryo-free portion of the seed was ground to produce a fine powder. 10-30 mg of seed powder was resuspended in a buffer containing 100 mM sodium phosphate (pH 7.5), 0.5 mM EDTA, and 0.5% Triton X-100, placed in a deep-well extraction block containing a 4 mm steel ball, and extracted in a Klecko homogenizer at maximum output for 45 seconds at room temperature. The block was centrifuged at 3000 × g for 15 minutes, and the supernatant was recovered. Protein was determined by the Bradford method, and keratinase activity was determined using a keratin-azure standard. Seeds were screened for the presence of transgenes using an internal fragment of the genotype Q53521 and the selectable marker PMI.

Replicate assays were performed according to the protease assay developed by Bressollier et al. (1999). Mechanically ground seed samples were incubated with 4 mg of a keratin standard (Sigma Aldrich) substrate in 1 mL of 50 mM Tris-HCl buffer (pH 7.5) at 50°C for 3 hours with constant agitation at 200 rpm. Five T1 seeds were used for each event. One unit of protease activity was defined as the amount of enzyme that resulted in a 0.01 U increase in absorbance at 595 nm (A595) after a 1-hour reaction with the keratin standard. Trypsin was used to estimate background activity. Figure 1 shows the determined enzyme activity of at least one progeny from each event, with replicates varying by ≤10%. No activity was detected in the AxB control and the trypsin control. These results are consistent with the expression of functional, activated keratinase from the pAG2209 expression cassette.

Example 3 Alternative strategy for the preparation of keratinase: first express the two inactive parts of keratinase, and then use trans-splicing inteins to promote the formation of functional protease

For the development of a successful cis-splicing protease, the solubility of the two parts of the intein-modified protease is advantageous for the association and efficient splicing of the two parts (Yamazaki, T. et al., 1998, Otomo et al., 1999). Similar to cis-splicing, trans-splicing is context-dependent and may require the addition of additional intein side chain residues at the insertion site.

A trans-spliced intein that may be present in intein-modified proteases or used in the methods herein is derived from Synechocystis sp. (Ssp) PCC6803 and is referred to as the trans-spliced intein for Ssp DnaE. In Synechocystis sp. (Ssp) PCC6803, the catalytic subunits of the duplicated DNA polymerase genes are encoded by two open reading frames separated by more than 700 kb on opposite chromosome strands. The trans-spliced Ssp DnaE intein assembles a functional protein from its two halves post-translationally. These two halves of the intein may be used for heterologous expression to assemble and splice intein-modified proteases and other proteins.

Examples of functional proteins assembled in plants from their inactive parts include: reconstruction from two parts of the functional β-glucuronidase gene (GUS) in Arabidopsis (Yang J. et al., 2003), glyphosate herbicide resistance from the genetic engineering of split 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) (Chin, HG et al., 2003), or sulfonylurea resistance recombined from the acetolactate synthase (ALS) protein assembled mediated by the DnaE intein from rice (Kempe et al., 2009). Cytotoxic barnase is also expressed as two inactive parts and assembled into an active protein by trans-splicing of the Ssp DnaB intein (Kempe et al., 2009). However, proteases have never been regulated or assembled using cis- or trans-spliced inteins. Unlike other enzymes that have been prepared by cis-splicing, proteases have different applications and can become active under different conditions compared to the other proteins mentioned that prefer spontaneous splicing over controlled splicing. If protease splicing occurs spontaneously within the host, the effect of the protease on the host will not be different from expressing a fully active protein (which is generally detrimental to the growth and development of the host). In contrast, for intein-modified EPSPS and ALS proteins, becoming active by spontaneous splicing is important in order to provide the host plant with tolerance to herbicides. Thus, EPSPS and ALS are beneficial to the plant host, while proteases are disadvantageous to the expression host, but are advantageous in subsequent applications of the protease, such as animal feed or laundry detergents.

Example 4 Selection of intein insertion sites in Q53521

To engineer the Q53521 protease with inteins, molecular modeling was used to identify intein insertion sites within the Q53521 protease. Three different approaches were used to select intein insertion sites.

1) The first approach is based on the need to activate the folding of the protease domain using a protease inhibitor prodomain. Cutting the enzyme between the two domains can inactivate the catalytic domain until the enzyme is spliced together and refolded. One site used here is the T108 site located between the domains.

2) The second approach used was to identify sites that are surface-exposed, near the ends of the secondary structure, or between the catalytic residues (D137, H168, S325) but near the ends of the domain. These conditions were chosen so that insertion of the intein into the enzyme would separate the active site residues but still allow for the most intrinsic contacts to partially promote pre-splicing folding of the protein. Sites were selected using the crystal structures 1 and 6 of chain A. These sites were S154 and T317.

3) Finally, the sites that were given the highest SVM splice cassette prediction scores were selected, which were located within or between the catalytic residues. See James Apgar, Mary Ross, Xiao Zuo, Sarah Dohle, Derek Sturtevant, Binzhang Shen, Humberto dela Vega, Phillip Lessard, Gabor Lazar, R. Michael Raab, "Predictive Model of Intein Insertion Site for Use in the Engineering of Molecular Switches," PLoS ONE, 7(5):e37355, 2012; DOI: 10,1371/journal.pone.0037355 and U.S. patent application Ser. No. 12/590,444, filed Nov. 6, 2009, all of which are incorporated herein by reference as if fully set forth. These sites were S234, S260, and S263. Other insertion sites were also attempted.

Example 5 Selection of intein

The intein of the trans-splicing of Synechocystis PCC6803DnaE is selected to build the intein-modified extein of Q53521. The nucleotide sequence and amino acid sequence of the N-terminal and C-terminal parts of the SspDnaE intein are codon-optimized for plant expression. The N-terminal and C-terminal parts of the intein are connected, and SspDnaE is synthesized into a single open reading frame (Codon Devices Co., Ltd.). By site-directed silent mutagenesis, the ctcgag sequence is converted to ctggag thereby internal XhoI site is removed. The underlined nucleotide sequence of the SspDnaE N-terminal with the ctggag sequence is as follows:

tgcctttctttcggaactgagatccttaccgttgagtacggaccacttcctattggtaagatcgtttctgaggaaattaactgctcagtgtactctgttgatccagaaggaagagtttacactcaggctatcgcacaatggcacgataggggtgaacaagaggtt ctggag tacgagcttgaagatggatccgttattcgtgctacctctgaccatagattcttgactacagattatcagcttctcgctatcgaggaaatctttgctaggcaacttgatctccttactttggagaacatcaagcagacagaagaggctcttgacaaccacagacttccattccctttgctcgatgctggaaccatcaag(SEQID NO:42)

Nucleotide sequence of the C-terminus of Ssp DnaE:

tggttaaggtgattggaagacgttctcttggtgttcaaaggatcttcgatatcggattgccacaagaccacaactttcttctcgctaatggtgccatcgctgccaat (SEQ ID NO: 43)

The amino acid sequence of the N-terminus of Ssp DnaE:

CLSFGTEILTVEYGPLPIGKIVSEEINCSVYSVDPEGRVYTQAIAQWHDRGEQEVLEYELEDGSVIRATSDHRFLTTDYQLLAIEEIFARQLDLLTLENIKQTEEALDNHRLPFPLLDAGTIK(SEQ ID NO:38)

Amino acid sequence of the C-terminus of Ssp DnaE:

MVKVIGRRSLGVQRIFDIGLPQDHNFLLANGAIAAN(SEQ ID NO:39)

Example 6 Expression of pro-Q53521 zymogen by λ phage Construction and expression of trans-splicing pairs of pro-Q53521-DnaE-N and DnaE-C-Q53 from λ phage

To test whether trans-splicing is feasible, six pairs of expression cassettes were prepared for co-expression in E. coli. The N-terminal domain of Q53521 was covalently fused to the N-terminal portion of the DnaE segmented intein (NI).

Bacteriophage lambda is used to express intein-modified enzymes within phage plaques and to screen for trans-splicing-regulated enzyme activity on diagnostic agar plates. These plates contain a colorimetric substrate that turns blue in the presence of active proteases. This system is suitable for expressing pro-Q53521. Furthermore, the trans-spliced Q53521-DnaE extein pair NI and IC was expressed in phage plaques and phage lysates. Protein expression was monitored by SDS/PAGE, and recovery of protease activity was tested in vitro on protease diagnostic plates.

The internal XhoI site was removed from the maize codon-optimized Q53521 and the precursor enzyme pro-Q53521 was cloned into the EcoRI and XhoI sites of the bicistronic expression cassette of λUni ZAP XR (Agilent).

Protease diagnostic plates were set up to detect active phage-expressed pro-Q53521. NZY top agar (Stratagene manual for Uni ZAP XR) was supplemented with IPTG (2.5 mM) and 0.5% AZCL-casein (Megazyme). The plates were incubated overnight at 37°C until confluent lysis occurred and further incubated at 50°C for 6 hours.

Protease diagnostic gel diffusion assay plates (Sokol et al., 1979) were set up according to Zhao et al. (2004), except that 0.4% AZCL-casein (Megazyme) was used instead of casein to improve detection sensitivity. Protease activity was verified using commercial Bacillus licheniformis protease (Sigma P8038). The blue color development indicates protease activity. Detection sensitivity was 30 ng of protease in 90 minutes at 37°C. A negative control, blank vector, showed no blue color development.

The trans-spliced Ssp.DnaE intein was inserted into six sites of pro-Q53521: T108, S154, S234, S260, S263, and T317. Two constructs, NI and IC, were generated at each site to split Q53-DnaE. The constructs were generated by overlapping PCR and cloned into the EcoRI/XhoI sites of the λ Uni ZAP vector downstream of the bicistronic expression cassette to the lac promoter. Recombinant λ phage DNA was packaged into phage and processed according to standard procedures (Uni ZAP XR, Stratagene manual), but the plates were prepared as protease diagnostic plates.

The amino acid sequences of the six trans-splicing pairs of Q53:DnaE are listed in Trans-splicing Pairs NI-(1-6) and IC-(1-6). Pro-Q53521 separates into N and C fragments at six sites: T108, S154, S234, S260, S263, and T317. The Q53N fragment was fused in frame with the Ssp DnaE-N fragment to generate the NI, and the DnaE-C fragment was fused in frame with the Q53-C fragment to generate the IC. The Ssp DnaE portion of the sequence is underlined and in bold. Molecular weight calculations were performed using the Compute pI/MW tool at www.expasy.org.

NI-1 Q53521-T108:DnaE-N(22.61Kd)(SEQ ID NO:13):

MAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQ *

IC-1 DnaE-C:T108-Q53521-C(31.1Kd)(SEQ ID NO:14):

TVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAV DNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMVSPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ*

NI-2 Q53521-S154:DnaE-N(27.25Kd)(SEQ ID NO:15):

MAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGA*

IC-2 DnaE-C:S154-Q53521-C(26.46Kd)(SEQ ID NO:16):

SFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNT NTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMVSPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ*

NI-3 Q52521-S234:DnaE-N(35.04Kd)(SEQ ID NO:17):

MAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANV KVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGA*

IC-3 DnaE-C:S234-Q53521-C(18.68 Kd)(SEQ ID NO:18):

SGSTAMKQAVDNAYARGVVVVAAAGNSGSSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMVSPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ*

NI-4 Q53521-S260:DnaE-N(37.53 Kd)(SEQ ID NO:19):

MAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASH PDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGN*

IC-4 DnaE-C:S260-Q53521-C(16.19 Kd)(SEQ ID NO:20):

NCSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMVSPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ*

NI-5 Q53521-S263-DnaE-N(37.76 Kd)(SEQ ID NO:21):

MAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHP DLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGS*

IC-5 DnaE-C:S263-Q53521-C(15.95 Kd)(SEQ ID NO:22):

NCSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMVSPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ*

NI-6 Q53521-T317:DnaE-N(43.20 Kd)(SEQ ID NO:23):

MAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHV AGTVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTN*

IC-6 DnaE-C:T317-Q53521-C(10.51Kd)(SEQ ID NO:24):

TYATLNGTSMVSPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ*

The protease activity of phage-expressed pro-Q53521 was tested using protease diagnostic plates. Trans-splicing was tested in sandwich plates of protease diagnostic plates of NI and IC trans-splicing pairs transfected with bacterial hosts.

Example 7 Expression of pro-Q53521 in Escherichia coli

Expression of pro-Q53521 in E. coli was performed according to the protocol of Tiwary and Gupta (2010), and the trans-splicing activity of the Q53521-DnaE NI and IC pairs co-expressed from the E. coli pETDuet-1 vector was evaluated.

Two sets of protease assays were set up: the AZO-casein method according to Radha and Gunasekaran (2008) and the QuantiCleave protease assay from Pierce (1992).

Pro-Q53521 was cloned into the XbaI and XhoI sites of the pET30b(+) vector (Novagen) with or without a C-terminal 6xHis tag, and the precursor keratinase was expressed using BL21(DE3)pLysS cells (E. coli). The starter culture was inoculated into an induction culture (4% v/v) in LB medium supplemented with 50 mg/L kanamycin and grown at 37°C at 300 rpm to an OD ₆₀₀ of 0.8. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM, and the culture was incubated for an additional 180 minutes. Aliquots were taken at time points 0, 30, 60, 90, 120, and 180 minutes. An 180-minute aliquot was separated into soluble (S) and insoluble (P) fractions: cells were harvested and lysed in 50 mM Tris (pH 7.5) supplemented with 1× Fastbreak (Promega) and 0.02 μl/ml Benzonase (Novagen) at room temperature for 30 minutes. The lysate was pelleted at 13K for 10 minutes. The supernatant was the soluble fraction (S), and the pellet was the insoluble fraction (P). Prokeratinase accumulated in the insoluble fraction. IPTG-induced pro-Q53521 was readily identified in SDS/PAGE stained with Coomassie Brilliant Blue for total protein.

The same pro-Q53521 expression vector, pET30b(+), was used to test alternative bacterial hosts: C3030H, Origami2(DE3)pLysS, BL21(DE3), and BL21star(DE3)pLysS. IPTG-induced accumulation of pro-Q53521 was readily detected in BL21(DE3), C3030, and BL21(DE3)pLysS. A starter culture was used to inoculate the induction medium (5% v/v) and grown in LB medium supplemented with the appropriate antibiotics at 25°C and 300 rpm to an _OD600 of ~0.7. IPTG was added to a final concentration of 0.1 mM. Aliquots at 0, 90, and 180 minutes were separated on a standard XT 12% Bis-Tris SDS/PAGE and stained with Coomassie Brilliant Blue using Simply Blue Safe Stain. The 0 and 90 minute samples were a 1:1 mixture of aliquots of the whole culture and 2× SDS loading dye. The 180 minute aliquot was separated into soluble (S) and insoluble (P) fractions: cells were harvested and lysed in 50 mM Tris, pH 7.5, supplemented with 1× Fastbreak (Promega) and 0.02 μl/ml Benzonase (Novagen) for 30 minutes at room temperature. The lysate was pelleted at 13K for 10 minutes. The supernatant was the soluble fraction, and the pellet was the insoluble fraction. Expression was supported by three host cells: BL21(DE3), BL21(DE3)pLysS, and C3030H, but the keratinase accumulated in the insoluble fraction in each host.

To facilitate analysis, traditional E. coli-based expression will be switched to expression in alternative hosts. For example, Bacillus subtilis and/or Pichia pastoris can be used, as both organisms are known to produce functional keratinase from Bacillus licheniformis PWD-1 (Lin et al., 1997; Wang and Shih 1999; Wang et al., 2003; Wang et al., 2004; Porres et al., 2002). Expression in Bacillus subtilis is also attractive due to its excellent secretion capacity, rapid growth, ease of manipulation, and the fact that it is a non-pathogenic bacterium with no endotoxins (Yeh et al., 2007).

Example 8 Conditioned Protease for the Laundry Detergent Industry

A major problem in the laundry industry is the instability of enzymes in detergents during storage. The stability problem of detergent enzymes is mainly due in part to the fact that the activity of detergent proteases can digest themselves and other detergent enzymes including proteases, lipases, amylases, cellulases, mannanases, xylanases and other enzymes.

The development of detergent proteases with modulated activity, which are inactive in a blend formulation but are activated during the wash cycle by detergent dilution, exposure to cold water, or a variety of other means, is an attractive goal. Proteases used in detergents, such as the subtilase Savinase, a subtilisin family alkaline protease, have been modified with inteins; however, this technology is also applicable to other subtilases (Siezen et al., 1997), which constitute an important class of proteases used in the detergent industry. Savinase (EC 3.4.21.62) is from Bacillus lentus and has been variously described as AprB peptidase (Bacillus B001), Esperase, Maxacal, Protease PB92 (Bacillus), Savinase, Super Savinase, Super 16L Savinase, Subtilisin 309, Subtilisin (a variant of Bacillus lentus), Subtilisin BL, or Subtilisin MC3. The amino acid sequences of Savinase and representative subtilisins include those set forth in SEQ ID NOs: 1-12.

The strategy for regulating Savinase is based on intein technology. Initially, it was envisioned to develop proteases modified with inteins that could be induced by cold splicing and/or by dilute detergents. Both cis- and trans-splicing inteins may be equally useful for the development of detergent enzymes.

Example 9 Expression of protease in E. coli system

Expression of Secreted Savinase in Bacillus subtilis WB800N

The nucleotide sequences encoding the Savinase catalytic domain of subtilisin preproSavinase, proSavinase, and Savinase P29600 (UniProt) were synthesized and cloned into the pUC57 (GenScript) plasmid. In the following amino acid sequences, the amino acid sequence of the signal peptide (residues 1-22) at the N-terminus is marked in bold, the prodomain is underlined, and the 269 amino acids of the catalytic domain are unmarked.

The sequence of Pre-pro Savinase, including the pre-signal peptide for secretion, is (SEQ ID NO: 1):

AEEAKEKYLIGFNEQEAVSEFVEQVEANDEVAI LSEEEEVEIELLHEFETIPVLSVELSPEDVDALELDPAISYIEEDAEVTTM AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQ AVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR.

The sequence of Pro-Savinase, including the pro-signal peptide for maturation, is (SEQ ID NO: 57):

AEEAKEKYLIGFNEQEAVSEFVEQVEANDEVAILSEEEEVEIELLHEFETIPVLSVELSPEDVDAL ELDPAISYIEEDAEVTTM AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQ AVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR.

The sequence of the Savinase catalytic domain is (SEQ ID NO: 58):

M AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLE QAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR

To test secretory expression of Savinase in Bacillus subtilis, the full-length protein (pre-pro-Savinase), pro-Savinase, and the catalytic domain of Savinase were cloned between the BamHI and AatII sites of pHT01 and pHT43 vectors with or without a C-terminal His tag from MoBiTec. These were expressed in Bacillus subtilis WB800N cells deficient in eight extracellular proteases. The sequences of the resulting constructs are listed in Table 4.

Table 4 Sequence numbers (SEQ ID NOs) of the nucleotide and amino acid sequences of the Savinase expression cassettes in the Bacillus subtilis vectors pHT01 and PHT43.

Construct Nucleic acid sequence number Amino acid sequence number pHT01-preproSavinase/P29600 59 1 pHT01-preproSavinase-8His 60 61 pHT01-proSavinase-8His 62 63 pHT01-Savinase-8His 64 65 pHT43-preproSavinase-8His 66 67 pHT43-proSavinase-8His 68 69 pHT43-Savinase-8His 70 71

pHT01 is a cytoplasmic expression vector, but secretory expression of the full-length Savinase protein is possible via the native secretion signal. pHT43 is a secretion vector with an N-terminal SamyQ secretion signal to direct the recombinant protein into the culture medium. In pHT43-preproSav-8His, the SamyQ secretion signal is followed by a stop codon, and the full-length protein with the native secretion signal is expressed from a bicistronic expression cassette. In pHT43-proSav-8His, the proenzyme is expressed along with a vector-encoded N-terminal SamyQ secretion signal. Bacillus subtilis treatment was performed according to MoBiTec protocols, except that transformation was performed according to Lu et al. (2012).

Savinase protein activity secreted by Bacillus subtilis WB800N cells was assayed on LB agar plates supplemented with 10 mM calcium chloride, 10 μg/ml chloramphenicol, 1 mM IPTG, and 0.25% AZCL-casein (Megazyme). Four biological replicates of Bacillus subtilis expressing each construct were plated on the agar plates and incubated overnight at 37°C. The release of blue dye surrounding bacterial growth indicated protease activity. Protease activity was detected in preproSavinase expression cassettes with or without an 8×His tag in the pHT01 and pHT43 vectors, as well as in the pHT43-SamyQproSav (no His-tag) construct, in which the native secretion signal was replaced with the SamyQ signal peptide.

The protease activity of savinase secreted from Bacillus subtilis supernatants was determined (Figure 2). Bacillus subtilis cultures were grown overnight at 37°C and 300 rpm in LB medium supplemented with chloramphenicol (10 μg/ml) and IPTG (1 mM). Two hundred μL of the culture was precipitated in 16K RCF for 5 minutes, and the supernatant was collected and diluted 1:1 with the same buffer containing 0.1 M Tris (pH 8), 0.5 mM calcium chloride, and an equal volume of 1% AZO-casein (Megazyme). The supernatant was incubated at 37°C for 30 minutes. The protein was precipitated by adding an equal volume of 5% TCA and allowed to stand at ambient temperature for 5 minutes. The precipitate was pelleted in a microcentrifuge at 16,000 RFC for 5 minutes. 100 μL of the supernatant was combined with 100 μL of NaOH, and the absorbance was read at 420 nm. Protease activity was measured in cultures expressing proSavinase with both a native secretion signal (pHT01) and a SamyQ secretion peptide (pHT43). Figure 2 shows the Savinase activity detected in suspension culture supernatants of preproSav with or without an 8×His tag (expression vectors pHT01 and pHT43), as well as in suspension culture supernatants of pHT43 and SamyQ proSav without a His tag. No activity was detected from the pre-Savinase in the cytoplasm (pHT01-proSav-His) or when the Savinase catalytic domain was expressed with either the native or SamyQ secretion signal.

The results were confirmed by Western blot analysis of Savinase expressed by Bacillus subtilis. Briefly, Bacillus subtilis cultures were grown overnight at 37°C at 300 rpm in LB medium supplemented with chloramphenicol (10 μg/ml) and IPTG (1 mM). Two hundred μL of culture were pelleted for 5 minutes in 16K RCF, and the supernatant was removed. The pellet was resuspended in 2× Laemmli buffer (BioRad) containing 5% β-mercaptoethanol at 1/10 the original culture volume, and an equal volume of water was added. The supernatant was mixed with an equal volume of 2× Laemmli buffer containing 5% β-mercaptoethanol, and before loading onto a 12% Bis-Tris polyacrylamide gel (BioRad), the supernatant and pelleted sample were boiled at 95°C for 5 minutes, and the gel was run at 150-160V. For Western blotting, proteins were transferred to a PVDF membrane and developed with the primary antibody "THE anti-His" (GenScript) and the secondary antibody HRP:: goat anti-mouse (Sigma) with HRP anti-biotin (Cell Signaling Technologies) to visualize molecular weight markers. The 28 kDa band represents mature Savinase. Secreted proSavinase+8His undergoes adequate maturation. ProSavinase expressed in the cytoplasm is not maturated (pHT01-proSav-8His). Enzyme activity correlates well with the accumulation of a ~28 kDa protein corresponding to mature Savinase in the supernatant. This lack of activity may be due to the fact that proSavinase is not maturated in the cytoplasm (pHT01-ProSav-8His) and that the Savinase catalytic domain is unstable in the cytoplasm and in the secreted form (pHT01-SAV-8His and pHT43-SamyQSav-8His).

These observations indicate that expression of active Savinase in B. subtilis requires secretion of proSavinase for proper maturation of the catalyzed active protease.

Expression of secreted Savinase in Escherichia coli

To test whether E. coli is a suitable host for secretory expression of Savinase, the nucleotide sequences of pro-Savinase and the catalytic domain of Savinase were cloned into the pET22b(+) secretion vector in frame with the N-terminal pelB signal peptide, and the pET22b-pelBproSav-6His and pET22b-pelBSav-6His vectors were created. The vectors were transformed into BL21(DE3) and Lemo21(DE3) E. coli strains. To test Savinase expression, overnight suspension cultures were pelleted and cells were separated into periplasmic and protoplast fractions using the Peripreps Periplasting Kit (PS81100) from Epicentre Biotechnologies. Samples were analyzed by Western blot, and protease activity was determined (Figures 3 and 4).

Briefly, the nucleotide sequence encoding the Savinase catalytic domain and pro-Savinase was cloned into the pET22b(+) secretion vector in frame with the N-terminal pelB signal peptide, creating pET22b-pelBSav-6His and pET22b-pelB-proSav-6His. The vectors were transformed into BL21(DE3) and Lemo21(DE3) E. coli strains, and suspension cultures were grown overnight at 30°C and 300 rpm in overnight instant expression TB medium (AIM, Novagen) supplemented with 100 mg/L carbenicillin. One milliliter aliquots were used to prepare periplasmic (P) and protoplast fractions (S) using the Peripreps Periplasting Kit (PS81100) from Epicentre Biotechnologies. For Western blot analysis ( FIG3B ), 5 μL of periplasmic and protoplast protein fractions were separated in a 4-12% gradient SDS/PAGE, blotted onto a PVDF membrane, and developed using a mouse anti-His tag primary antibody, goat anti-mouse HRP secondary antibody, and anti-biotin HRP for detection of biotinylated protein bands, and a Super Signal West Picowas (Pierce) for signal detection. FIG3 shows that a 28 kDa protein corresponds to mature Savinase in the periplasmic and protoplast fractions from both vectors and in both bacterial hosts. The observed accumulation of a band near 28 kDa in E. coli expressing pro-Savinase is consistent with pro-Savinase maturing into a catalytically active protein.

Figure 3A shows the activity of the Savinase enzyme. To assess activity, 50 μL aliquots of the periplasmic and protoplast protein fractions were subjected to a protease activity assay using the previously described method. Figure 3A shows that expression of proSavinase in both E. coli hosts produces an active protease, while expression of the Savinase catalytic domain produces an inactive protein. The protease activity assay demonstrated activity of the proSavinase expression cassette in both periplasmic and protoplast fractions of the two E. coli hosts. These observations are consistent with the results that proSavinase properly matures into catalytically active Savinase in E. coli. Expression of the catalytic domain alone results in an inactive protein. This expression-activity curve demonstrates that the prodomain plays a role in the maturation of active Savinase.

Cytoplasmic Expression of Savinase in Escherichia coli

To test Savinase expressed in the cytoplasm of E. coli, the nucleotide sequences of the full-length proteins preproSavinase, proSavinase, and the catalytic domain of Savinase were cloned into the EcoRI/XhoI sites of pBluescript II XR (Agilent) and expressed in E. coli SOLR cells (Stratagene). Savinase activity was determined from overnight cultures grown at 30°C and 300 rpm in 5 mL overnight Instant Expression TB medium (AIM, Novagen) supplemented with 100 mg/L carbenicillin and 0.25 mM IPTG. Cells were harvested by centrifugation at 3000 rpm for 10 minutes at 4°C, and the pellet was dissolved in poly-buffer (pH 6.5) containing 100 μL of Fastbreak (1×) for 60 minutes, followed by the addition of 400 μL of poly-buffer. To determine enzyme activity, 100 μL of the lysate was added to 100 μL of 0.1 M Tris-HCl pH 8.0 containing 1% AZO-casein and 0.5 mM CaCl ₂ , and then incubated at 55° C. for 30 minutes. The reaction was terminated by adding 200 μL of 5% (w/v) trichloroacetic acid, pelleted at 5000 rpm for 5 minutes, and the absorbance of the supernatant was measured at 340 nm. Protease activity was detected only from the proSavinase expression cassette. Figures 4A-4B show the expression of pre-proSavinase, proSavinase, and the Savinase catalytic domain in E. coli SOLR cells using pBluescript.

Fig. 4 A shows the enzymatic activity assessment of the cell lysates of the Escherichia coli SOLR cells expressing full-length proteins preproSavinase, proSavinase and Savinase catalytic domain (Savinase). Mean value and standard deviation are based on three biological replicates. By observation, only activity can be detected from the proSavinase expression cassette, while all inactivity is absent when expressing the full-length protein or expressing the catalytic domain separately.

The effect of Savinase activity on the growth of E. coli SOLR cells was evaluated (Figure 4B). E. coli SOLR expressing preproSavinase, proSavinase, and the Savinase catalytic domain were inoculated into 5 mL of overnight instant expression TB medium (AIM, Novagen) supplemented with carbenicillin (100 mg/L) and cultured at 37°C for 10 hours, followed by culture at 30°C for 6 hours. The absorbance of 500 μL of culture was measured at 590 nm. E. coli SOLR cells expressing active pro-Savinase grew poorly. As shown in Figure 4B, it was observed that cytoplasmically expressed proSavinas slowed growth, indicating that the protease activity was harmful to the cells. Similarly, the cytotoxic effect of proSavinase was also found in other E. coli cells (Top10, DH5α, and BL21) and in yeast that was found to be cold-induced in the high-throughput screening of proSavinase.

The strategy described herein for regulating Savinase is based on intein technology. It is desirable to develop an intein-modified protease that can be induced to splice by cold and/or dilute detergents. Both cis- and trans-splicing inteins are also useful in the development of detergent enzymes.

Example 10 Strategies for regulating protease activity

Intein technology is being used to develop enzymes whose activity can be precisely controlled for specific applications, for example, to create intein-modified proteases (iProteases) whose activity can be modulated by varying the concentration of detergent formulations. The goal of modulating protease activity is to improve the stability of proteases in liquid detergents used in household care products. Eleven subtilisins (SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11) were analyzed, and proSavinase (SEQ ID NO: 57) was used for intein modification. Functional cis- and trans-splicing of the intein-modified proteases was obtained, induced by lowering the solution temperature or by diluting the concentrated detergent formulation. Furthermore, the intein-modified Savinase was shown to effectively remove stains through intein splicing, as measured by a stain removal assay.

Cis- and trans-splicing inteins at various Savinase insertion sites were evaluated, and the effects of regulating intein splicing in response to cold and detergent dilution were investigated. Analysis of various molecules and inducing stimuli demonstrated that detergent-induced splicing was the most effective modulator of Savinase activity. The key indicator achieved for the primary trans-splicing molecules (iSavinase-S317:Gp41-1 NI and IC) was post-splicing activity after a 125-fold dilution of detergent formulated with the iSavinase-S317:Gp41-1 trans-splicing protease into water, compared to virtually zero activity (incapable of accurate measurement above baseline) when the iSavinase-S317:Gp41-1 NI and IC were tested in detergent alone or after a 125-fold dilution into detergent. The activity difference demonstrated by this molecule in the dilution assay of the present invention was stable for over 17 days, whereas unmodified Savinase lost all activity within five days using the same method. Furthermore, the molecule exhibited significant stain removal capabilities when 20 μL to 100 μL of harvested protein was loaded onto fabric disks contaminated with blood, or a mixture of blood, milk, and ink.

In addition to the development of dilution-regulated trans-splicing, savinase molecules that are induced by dilution of detergent or by exposure to lower temperatures (20°C) were also developed. The expression systems tested were: Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, phage, and in vitro transcription and translation (IVTT). Dilution- and cold-inducible screening systems, as well as novel assays for detecting enzyme activity during formulation, were developed.

The intein-modified Savinase molecule is referred to herein as "iSavinase," and the intein-modified precursor molecule expressed prior to intein splicing is referred to herein as "NIC" (denoting amino-extein (N) fused to carboxy-extein (C) in an intein (I)). Depending on the molecule being developed, i.e., whether a cis- or trans-splicing intein is used, and the desired stimulus for modulating splicing, various approaches are selected in order to minimize development time while still optimizing the desired properties of the molecule. Regardless of the system used, two types of assays are commonly used to develop modulated activities. The first type of assay is referred to as an "inhibition" assay and is used to screen intein-modified molecules for reduced activity under conditions targeted to inhibit intein splicing and, further, to inhibit enzyme activity ( FIG5 ).

Figure 5 shows the inhibition assay developed for unmodified (Savinase) and intein-modified Savinase (iSavinase). Referring to this figure, Savinase and iSavinase molecules were analyzed under various conditions and regulatory stimuli to determine differences in relative activity. The activity of Savinase and iSavinases was measured across a series of detergent concentration gradients compared to water to determine the concentration at which intein-modified Savinase was selectively less active. Similarly, the same molecules were exposed to different temperatures to determine the relative impact of temperature exposure on intein splicing.

Figure 6 illustrates a second type of assay, known as an "induction" assay, which is used to screen intein-modified molecules for activity recovery following exposure to splicing stimuli such as temperature or detergent dilution. While inhibition assays help identify molecules with the desired level of activity, induction assays address the question of whether inhibition is reversible and whether activity can be recovered from the inhibited state. Induction assays can more accurately represent how these molecules will be used in home care products. Both assays facilitate the development of regulated intein-modified proteases with the desired level of activity. Referring to Figure 6, in an induction assay, unmodified (Savinase) and intein-modified Savinase (iSavinase) molecules are initially exposed to the desired inhibitory conditions to measure their activity. Thereafter, the molecules are exposed to activating conditions or removed from the inhibitory conditions, whereupon their activity is measured again and compared to intein splicing to determine the amount of activity recovered. For example, the activity of unmodified and intein-modified Savinase enzymes is measured in a high-concentration detergent formulation. The formulated molecules are then diluted in water or a detergent of the same concentration. Intein-modified savinases that were activated upon dilution in water but not in detergent were selected for further development. Similarly, the same molecules were assayed by exposure to different temperatures to determine the relative effects of temperature exposure on intein splicing.

Devising strategies to pursue the development of regulated proteases requires addressing several significant technical challenges. In particular, the challenge of being able to separate the precursor, intein-modified protease from the expression conditions where it is desirable to have splicing of the molecule is an important consideration. That is, it is desirable to have splicing of the intein-modified protease at mild temperatures in a low detergent (aqueous) environment, which is the exact condition used to express heterologous proteins in most common host organisms (such as Bacillus, E. coli, or yeast).

Three design strategies were chosen to address this issue. The first strategy was to use a trans-splicing intein, in which the protease would be split into two inactive fragments that could be expressed separately and assembled after mixing. While trans-splicing inteins alleviates the challenges associated with preparing precursor molecules, regulated trans-splicing is a poorly studied phenomenon requiring additional engineering development. The second strategy was to utilize cold-induced splicing during preparation and as an alternative to dilution-induced splicing from high detergent concentrations. If low temperatures are required for intein splicing, the precursor molecule can be prepared, isolated, and formulated at elevated temperatures (typically 25°C to 42°C, depending on the expression system). Once formulated, the temperature can be lowered to ambient temperature, and until dilution occurs, the detergent formulation will inhibit splicing. The rationale for cold-induced splicing as an alternative to detergent dilution splicing was based on analyses of cold-induced splicing documented in the literature. Previously shown molecules splicing at lower temperatures showed that lower temperatures stabilize the intein, confirming that this facilitates intein splicing. This is in contrast to inteins, which have been shown to be unstable and largely inactive at higher temperatures. Likewise, such inteins are less stable in high concentrations of detergent, but become stable when the detergent is diluted and the temperature of the aqueous solution is at or below ambient temperature (<25°C). A third strategy is to screen for intein-modified proteases that are relatively insoluble when overexpressed, but can be isolated, solubilized by detergent, and refolded, but have low splicing activity until the detergent is diluted. This strategy is the riskiest one, as insoluble proteins are notoriously difficult to redissolve in an active form, let alone be modulated by the presence of detergent. The following examples describe preliminary testing of Savinase intein modifications and the results obtained using each strategy. Each example describes the expression system used, the detection method employed, and the primary candidate properties measured in the experiments.

Example 11 Selection of intein insertion sites in Savinase

Molecular modeling was used to select sites for cis-splicing inteins into the Savinase protease. Sites with wild-type serines or threonines were analyzed for their potential compatibility for intein insertion (wild-type Savinase lacks cysteine). Three approaches were used to select these sites:

[1] Considering the inhibitory effect of the pro-domain of the protease, a serine position (S114) was selected that is located close to the pro-domain cleavage site. The unspliced intein at this position can inhibit the cleavage of the pro-domain and thus activate the protease.

[2] The second approach is to identify sites near the protein surface that have similar characteristics to previously discovered successful intein insertion sites. Characteristics include solvent accessibility, secondary structure, local hydrogen bonding environment, residue identity, insertion in a homologous protein, proximity to the active site, and proximity to a protein terminus. See James Apgar, Mary Ross, Xiao Zuo, Sarah Dohle, Derek Sturtevant, Binzhang Shen, Humberto dela Vega, Phillip Lessard, Gabor Lazar, R. Michael Raab, “Predictive Model of Intein Insertion Site for Use in the Engineering of Molecular Switches,” PLoS ONE, 7(5):e37355, 2012; DOI: 10,1371/journal.pone.0037355, which is incorporated herein by reference as if fully set forth. Sites were selected using 1GCI chain A. The sites selected were T148, S166, S253, S269, and S347.

[3] Different intein insertion sites were experimentally tested by inserting intein coding sequences upstream of each serine or threonine in the Savinase coding sequence.

Due to the importance of intein splice sites, in addition to using the natural serines and threonines for intein insertion, the Savinase protein can be mutagenized to incorporate some or all of the wild-type intein splice cassette amino acids at any desired splice site in Savinase.

Example 12 Selection of intein

Three inteins were selected for preliminary testing of intein splicing in Savinase protease. First, the Saccharomyces cerevisiae vacuolar ATPase subunit (VMA) intein was chosen due to previous success with cold-temperature splicing. Second, the Thermus thermophilus HB27 DnaE-1 Tth intein and its engineered miniature mTth intein were selected due to previous successful splicing of this intein in other proteins. Third, inteins trans-spliced by SSP and DnaE were selected for the preparation of intein-modified proteases. The N-termini of these inteins were inserted into selected sites. Savinase, Tth, or mTth inteins with VMA were inserted into six or 20 selected sites, respectively, as listed in SEQ ID NOS 25-36, 73-92, and 120-139. Constructs containing proteases with intein sequences are also listed in Table 2.

Example 13 Mutagenesis to make any position available as an intein insertion site

A common requirement for intein splicing is the presence of a serine, threonine, or cysteine amino acid at the C-terminal binding site between the inserted intein and the carboxy-terminal extein. Given modern molecular biology techniques, any position in a protein can be mutated to contain a serine, threonine, or cysteine. Alternatively, using modern molecular biology techniques, a serine, threonine, or cysteine can be inserted between any two residues in a protein sequence. In either case, the N-terminus of the intein can be inserted with the modified amino acid and then used to test for conditional intein splicing.

Example 14 Expression of intein-modified subtilisin

Escherichia coli and Bacillus species are attractive systems for expressing proteases, which can be produced as secreted proteins or using intracellular expression (Phrommao et al., 2011). An alternative method is to secrete the recombinant protein into the culture medium; this approach has several advantages, including the ability to screen for enzyme activity in culture supernatants or on diagnostic agar plates containing a colorimetric substrate that changes color in the presence of active enzyme. Secretion of recombinant Bacillus hydrolases in an E. coli expression system (Yamabhai et al., 2008) demonstrated that various Bacillus signal peptides can be recognized by E. coli. Subtilisins have been successfully expressed in several expression systems, including E. coli (Phrommao et al., 2011; Fang et al., 2010), B. subtilis (Tindbaek et al., 2004; Pierce et al., 1992), and bacteriophage (Legendre et al., 2000). New research advances in Bacillus subtilis protein expression systems include commercially available E. coli-B. subtilis shuttle vectors for intracellular and secretory expression, B. subtilis expression hosts lacking eight extracellular proteases, and more efficient transformation methods that can produce up to 4 × 10 ⁵ transformants/μg DNA (Guoquiang et al., 2011), making B. subtilis an attractive host for the development of intein-modified proteases.

Mutated intein libraries can be generated by a variety of methods including random mutagenesis, targeted and saturation mutagenesis, chemical mutagenesis, domain shuffling, and overlapping PCR to reconstitute beneficial mutations. One method for inserting an intein library into the target protein Savinase is by overlapping PCR of three DNA fragments encoding the intein and Savinase's N-extein and C-extein flanking the insertion site. Another method is to linearize a vector containing the Savinase coding sequence and co-transform a vector containing an intein sequence library with overlapping 5'- and 3'-sequences with a restriction vector into yeast, in which yeast recombination directly assembles the intein-modified protease into the DNA vector. The gene encoding Savinase with intein inserted into the frame can be subcloned into an appropriate expression vector when necessary and the library can be transformed into an appropriate expression host. Savinase can be expressed in E. coli using the pET21d vector and the BL21 (DE3) host. For expression and screening in Bacillus subtilis, the mutant library was constructed into a shuttle vector in E. coli and then transformed into B. subtilis. Expression of intein-modified Savinase in E. coli or phage systems provides the additional advantage of allowing high-throughput screening of mutants from the mutagenesis library as described in Example 16.

Example 15 Determination of Savinase Enzyme

The substrate used for the Savinase enzyme assay is the chromogenic peptide substrate N-succinyl-alanyl-alanyl-proline-phenylalanine p-nitroanilide (N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide, Sigma-Aldrich). This substrate is highly specific for subtilisin enzymes (Davis et al., 1999) and supports enzyme assays in bacterial suspensions (Bonifait et al., 2010). In a typical test, 20 μL of the chromogenic substrate N-succinyl-alanyl-alanyl-proline-phenylalanine p-nitroanilide (2 mg/ml in 50% dimethylformamide) is added to 100 μL of lysate or bacterial suspension, the reaction mixture is incubated at 37 ° C for different times, and the released pNA is quantified by measuring the absorbance at 415 nm (Bonifait et al., 2010). This operating method is easily adapted to support screening by performing high-throughput protease activity assays through automation. Proteolytic activity can also be measured by digestion of AZO-casein (Vazquez et al., 2004). 20 μL of lysate and 20 μL of 1% (w/v) AZO-casein in Tris-HCl buffer (0.1 M, pH 8.0) and 0.5 mM calcium chloride were incubated in a 384-well plate at 55°C for 30 minutes. After terminating the reaction with 40 μL of 5% (w/v) trichloroacetic acid, the reaction mixture was centrifuged and the absorbance of the supernatant was measured at 340 nm.

Example 16 High Throughput Screening

Screening can be automated to support high-throughput enzyme assays (Bonifait et al., 2010); or when protease activity is detected in the region of the gap (when using phage) and when the phage or microbial host is used to release the pigment from the chromogenic substrate, diagnostic plates are used (Phrommao et al., 2011; You and Arnold, 1994). Screening can be achieved by exploiting the cytotoxicity of proteases to select cells expressing conditional splicing proteases and eliminate spontaneous splicing of those intein-modified proteases (see Example 23).

Example 17 Insertion of intein into proSavinase

mTth: EU59 recombinant intein inserted into 20 positions before the underlined amino acids of proSavinase after removal of the 26 amino acid pre-signal peptide: S46, S62, T77, S86, S100, T109, S135, T148, S166, T167, S196, S208, S239, T243, S269, T285, S293, S317, T318, T329 (SEQ ID NO: 57):

MAEEAKEKYLIGFNEQEAV S EFVEQVEANDEVAIL S EEEEVEIELLHEFE T IPVLSVEL S PEDVDALELDPAI S YIEEDAEV T TMAQSVPWGISRVQAPAAHNRGLTG S GVKVAVLDTGIS T HPDLNIRGGASFVPGEP ST QDGNGHGTHVAGTIAALNNSIGVLGVAP S AELYAVKVLGA S GSGSVSSIAQGLEWAGNNGMHVANLSLGSP S PSA T LEQAVNSATSRGVLVVAASGNSGAG S ISYPARYANAMAVGA T DQNNNRA S FSQYGAGLDIVAPGVNVQSTYPG ST YASLNGTSMA T PHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGSTNLYGSGLVNAEAATR.

The construct was cloned between the EcoRI and XhoI sites of pBluescript II XR (Agilent) using overlapping PCR and transformed into E. coli SOLR cells (Stratagene). The nucleotide sequence of the mTth:EU59 recombinant intein shown in SEQ ID NO:72, encoding the mTth:EU59 recombinant intein (SEQ ID NO: 119), was inserted into proSavinase, resulting in the production of the intein proSavinase constructs: proSavinase S46-mTth:EU59 (SEQ ID NO: 73), proSavinase S62-mTth:EU59 (SEQ ID NO: 74), proSavinase T77-mTth:EU59 (SEQ ID NO: 75), proSavinase S86-mTth:EU59 (SEQ ID NO: 76), proSavinase S100-mTth:EU59 (SEQ ID NO: 77), proSavinase T109-mTth:EU59 (SEQ ID NO: 78), proSavinase S135-mTth:EU59 (SEQ ID NO: 79), proSavinase T148-mTth:EU59 (SEQ ID NO: 80), proSavinase T160-mTth:EU59 (SEQ ID NO: 81), proSavinase T175-mTth:EU59 (SEQ ID NO: 82), proSavinase T180-mTth:EU59 (SEQ ID NO: 83), proSavinase T190-mTth:EU59 (SEQ ID NO: 84), proSavinase T201-mTth:EU59 (SEQ ID NO: 85), proSavinase T210-mTth:EU59 (SEQ ID NO: 86), proSavinase T225-mTth:EU59 (SEQ ID NO: 87), proSavinase T230-mTth:EU59 (SEQ ID NO: 88), proSavinase T240-mTth:EU59 (SEQ ID NO: 89), proSavinase T251-m proSavinase S239-mTth: EU59 (SEQ ID NO: 85), proSavinase T243-mTth: EU59 (SEQ ID NO: 86), proSavinase S269-mTth: EU59 (SEQ ID NO: 87), proSavinase T285mTth: EU59 (SEQ ID NO: 88), proSavinase S293mTth: EU59 (SEQ ID NO:89), proSavinase S317-mTth: EU59 (SEQ ID NO:90), proSavinase T318-mTth:EU59 (SEQ ID NO:91), and proSavinase T329-mTth:EU59 (SEQ ID NO:92) encoding the following intein-modified proSavinases: proSavinase S46-mTth:EU59 (SEQ ID NO:120), proSavinase S62-mT:EU59 (SEQ ID NO:121), proSavinase T77-mTth:EU59 (SEQ ID NO:75122), proSavinase S86-mTth:EU59 (SEQ ID NO:123), proSavinase S100-mTth:EU59 (SEQ ID NO:124), proSavinase T109-mTth:EU59 (SEQ ID NO:125), proSavinase S135-mTth:EU59 (SEQ ID NO:126). NO:126), proSavinaseT148mTth:EU59 (SEQ ID NO:127), proSavinase S166-mTth:EU59 (SEQ ID NO:128), proSavinase T167-mTth:EU59 (SEQ ID NO:129), proSavinase S196-mTth:EU59 (SEQ IDNO:130), proSavinase S208-mTth:EU59 (SEQ ID NO:131), proSavinase S239-mTth:EU59 (SEQ ID NO:132), proSavinase T243-mTth:EU59 (SEQ ID NO:133), proSavinase S269-mTth:EU59 (SEQ ID NO:134), proSavinase T285mTth:EU59 (SEQ ID NO:135), proSavinaseS293mTth:EU59 (SEQ ID NO: 136), proSavinase S317-mTth:EU59 (SEQ ID NO: 137), proSavinase T318-mTth:EU59 (SEQ ID NO: 138), and proSavinase T329-mTth:EU59 (SEQ ID NO: 139. Inteins were also inserted into proSavinase using yeast homologous recombination (see Example 23 Cold-regulated cis-splicing iSavinase).

Example 18 Intein insertion into proSavinase can inhibit Savinase activity and splicing can restore Savinase activity

E. coli SOLR cells expressing iproSavinases were grown in overnight cultures, and cell lysates were prepared as described in Figure 5A. To test for temperature-induced splicing, aliquots of the cell lysates were incubated at 4, 37, or 55°C for 2 hours. The proteins were then resolved by SDS/PAGE and Western blotted with an intein-specific antibody against EU59. Figure 7B shows a Western blot of intein splicing. The iproSavinase (NIC) site and free intein (mTth:EU59) released after splicing are labeled on the left. The intein insertion sites are shown below. The three lanes at each insertion site correspond to aliquots pretreated at 4, 37, and 55°C for 2 hours, respectively. Below the line, a (+) or (-) for each insertion site indicates the presence or absence of spliced free intein-derived xylanase activity. Intein splicing was scored by releasing inteins from the precursor, and 5 out of 20 insertion sites were detected, including S135, S269, S293, S317, and T318.

Intein splicing restores Savinase activity, as demonstrated by enzyme activity assays. E. coli SOLR cells expressing intein-modified proSavinase were cultured, and enzyme activity from bacterial lysates was assayed as shown in FIG5A .

Figures 7A-7B show the correlation between target enzyme activity and intein splicing. A modified mTth intein of xylanase EU59 was inserted into proSavinase at different serine (S) and threonine (T) sites to generate a multiprotein expression cassette expressing both a xylanase and a protease. For each construct, 16 biological replicates were tested: 8 at 37°C (filled rectangles) and 8 at 55°C (open rectangles) were pre-incubated for 2 hours to promote splicing and enzyme activity recovery prior to enzyme activity assays. Cell lysates from each expression cassette were assayed for enzyme activation and intein splicing. Intein-modified proSavinases that exhibited splicing also exhibited enzymatic activity (S135, S269, S293, and S317).

Figure 7A shows the Savinase activity of four of the seven expression cassettes (S135, S269, S293, and S317) demonstrated in a protease assay. Savinase activity from these expression cassettes was obtained after preheating at 37°C and 55°C for 2 hours. Figure 7B illustrates a Western blot using EU59 antiserum. Referring to this figure, a band matching the size of the EU59-modified mTth intein was detected for all four expression cassettes shown in Figure 7A. mTth:EU59 intein splicing was observed in the S135 and S317 expression cassettes at all three temperatures tested (dashed 4°C, thin line 37°C, and thick line 55°C, 2-hour treatment). However, in the S269 and S293 expression cassettes, intein splicing was only observed after preheating the lysate at 55°C. These results suggest that intein modification can be a useful tool for controlling protease activity.

Example 19 Savinase modified with mVMA:P77Cd and mTth:P77Cd

Similar to EU591743 (EU59), XynB (accession number P77853) is also a GH11 family xylanase. Its catalytic domain, P77853Cd (P77Cd) (SEQ ID NO: 714), shares sequence identity with the EU59 xylanase (SEQ ID NO: 715). Compared to full-length XynB, P77Cd expresses well in E. coli, is highly soluble in solution, and exhibits enhanced thermotolerance and specific activity. SceVMA is an intein that has been extensively studied and successfully used to develop cold-inducible protein switches. A homing endonuclease domain is predicted within its sequence.

P77Cd was internally fused to SceVMA, replacing the HEN domain. Four constructs were generated, with no link or an 8-amino acid link to the N-terminus, C-terminus, or both the C-terminus and the C-terminus of P77Cd. When expressed in E. coli, constructs with no link or a single link between P77Cd and SceVMA exhibited superior xylanase activity against the AZCL-xylan substrate, demonstrating that the modified intein (mVMA:P77Cd; SEQ ID NO:684) possesses xylanase activity. The nucleic acid sequence of SEQ ID NO:699 encodes the modified mVMA:P77Cd. The modified mVMA:P77Cd intein was inserted before S135, S265, S269, S293, S312, S317 and S326 by Savinase to generate the constructs iproSavS135:mVMA:P77Cd (SEQ ID NO: 701), iproSavS265:mVMA:P77Cd (SEQ ID NO: 702), iproSavS269:mVMA:P77Cd (SEQ ID NO: 703), iproSavS293:mVMA:P77Cd (SEQ ID NO: 704), iproSavS312:mVMA:P77Cd (SEQ ID NO: 705), iproSavS317:mVMA:P77Cd (SEQ ID NO: 706), iproSavS326:mVMA:P77Cd (SEQ ID NO: 707). For iproSavS312:mVMA:P77Cd and iproSavS326:mVMA:P77Cd, alanine mutations were also introduced at the ends of the inteins to form defective inteins with sequence numbers SEQ ID NO: 712 (iproSavS312:mVMA-C:P77Cd) and SEQ ID NO: 713 (iproSavS326:mVMA-C:P77Cd). The constructs encode proteins having the following amino acid sequences: iproSavS135:mVMA:P77Cd (SEQ ID NO: 686), iproSavS265:mVMA:P77Cd (SEQ ID NO: 687), iproSavS269:mVMA:P77Cd (SEQ ID NO: 688), iproSavS293:mVMA:P77Cd (SEQ ID NO: 689), iproSavS312:mVMA:P77Cd (SEQ ID NO: 690), iproSavS317:mVMA:P77Cd (SEQ ID NO: 691), iproSavS326:mVMA:P77Cd (SEQ ID NO: 692), iproSavS312:mVMA:P77Cd (SEQ ID NO: 693), iproSavS317:mVMA:P77Cd (SEQ ID NO: 694), iproSavS318:mVMA:P77Cd (SEQ ID NO: 695), iproSavS329:mVMA:P77Cd (SEQ ID NO: 696). =The mVMA:P77Cd intein was spliced using the mVMA:P77Cd intein (SEQ ID NO: 697) and iproSavS326:mVMA-C:P77Cd (SEQ ID NO: 698). Xylanase analysis indicated that the mVMA:P77Cd intein was capable of splicing, as high levels of xylanase activity were observed in iproSavS312:mVMA:P77Cd and iproSavS326:mVMA:P77Cd but not in their defective counterparts ( FIG8 ).

Similarly, an mTth:P77Cd intein was constructed by inserting P77Cd into the mTth intein. Four mTth:P77Cd constructs were generated by PCR, depending on the presence of a linker and whether it was present between the amino termini of P77Cd and mTth or between the carboxyl termini of P77Cd and mTth. These constructs were expressed in E. coli SOLR cells as described above. Xylanase activity assays showed that three of the four constructs (with the eight amino acids linked to the 3', 5', or both ends of P77Cd) produced greater than 50% of the xylanase activity of P77Cd. Constructs with 3' linkers inserted proSavinase at sites S135, S269, S293, and S317 to generate the following new constructs: iproSavS135:mTth:P77Cd (SEQ ID NO: 708), iproSavS269:mTth:P77Cd (SEQ ID NO: 709), iproSavS293:mTth:P77Cd (SEQ ID NO: 710), and iproSavS317:mTth:P77Cd (SEQ ID NO: 711).

To determine the xylanase activity of xylanase-modified inteins, such as mTth:P77Cd, E. coli SOLR cells expressing the modified intein were inoculated from a single colony and cultured in 96-well plates containing 1 ml of AIM (Novagen) supplemented with carbenicillin (100 mg/l) at 37°C for 10 hours, followed by 6 hours at 30°C in a shaking incubator (New Brunswick) at 900 rpm. Cells were harvested at 4000 rcf for 10 minutes, and the pellets were resuspended in 100 μL of lysis buffer containing 200 mM sodium phosphate (pH 6.5), 1× Fastbreak lysis buffer ^™ (Promega), and 0.2 μL of Benzonase nuclease/mL (Novagen). An additional 400 μL of 200 mM sodium phosphate buffer (pH 6.5) was added to each lysate. 70 μL of lysate was transferred to a 384-well plate, heat treated at 25°C to 65°C for 4 hours, and cooled to 25°C. All samples were mixed with 0.2% (w/v) finely ground solid AZCL-xylan oat (Megazyme) substrate and incubated at 37°C for approximately 1 hour. The reaction samples were vortexed, centrifuged at 4000 rcf for 7 minutes, and the absorbance of 50 μL aliquots of the supernatant was measured at 590 nm on a Paradigm microplate reader. The average activity and standard deviation were calculated from the analysis of extracts from 8-12 independently inoculated replicate cultures.

Figure 8 shows that xylanase assays demonstrate the utility of the mVMA:P77Cd and mTth:P77Cd inteins. Referring to the figure, mVMA:P77Cd and mTth:P77Cd exhibited xylanase activity with or without the 8-amino acid peptide linker inserted before P77Cd(L5') or after P77Cd(L3'). Xylanase activity was restored when mVMA:P77Cd was inserted with proSavinase at sites S312 and S326 (pSavS312:mVMA:P77Cd and pSavS326:mVMA:P77Cd, respectively). However, disabling intein splicing also abolished xylanase activity, demonstrating that these modified mVMA:P77Cd and mTth:P77Cd inteins can be spliced.

Example 20 Detergent Dilution-Induced Protease

Screening for intein-modified proteases induced by dilution presents a unique challenge: the intein-modified protease expressed prior to splicing must be able to formulate the protein into detergent but still allow splicing after detergent dilution, which may require conditions similar to those under which it was expressed. Therefore, the challenge is to express a stable intein-modified protease at low concentrations or in the absence of detergent, formulate the intein-modified protease in a detergent that prevents splicing, and then activate splicing upon dilution of the detergent. Several strategies can be used to address this challenge. One strategy is to define expression conditions that differ from those under which dilution of detergent occurs. For example, the intein-modified protease can be expressed at a higher temperature where splicing is inhibited, the detergent can be formulated at the inhibitory temperature, and then diluted at a lower temperature (<20°C) that triggers intein splicing and protease activation. Although detergents do not necessarily play a role in splicing under these conditions, their role in intein splicing may also be exploited. For example, splicing by an intein-modified protease can be identified only under conditions where the intein is exposed to certain dilutions of detergent, where concentrated detergent inhibits splicing.

Another strategy is to express the intein-modified protease in a form where splicing is inhibited, such as by solubilizing the intein-modified protease in a detergent that allows refolding but inhibits splicing at high concentrations where aggregation or inclusion body formation is likely to occur, and then diluting the refolded intein-modified protein in a detergent that allows splicing. A similar strategy can be used to express the intein-modified protease so that the enzyme is secreted into a matrix containing a splicing inhibitor (such as zinc), forming the intein-modified protease in the presence of the inhibitor and detergent, and then diluting the inhibitor and detergent to allow splicing and activation of the intein-modified protease. Another strategy is to use a trans-splicing intein-modified protease, in which two intein-modified exteins are expressed separately and formulated in a detergent that prevents splicing, but allows trans-splicing of the inteins upon dilution of the detergent to activate the protease.

Example 21 Dilution-regulated trans-splicing iSavinase

Trans-splicing inteins support two pathways for controlling Savinase activity. Figure 9 shows the assembly of a trans-splicing protein. Referring to the figure, Savinase is split into two inactive peptide fragments, which are expressed separately as fused to trans-splicing inteins, amino-intein (NI) and carboxyl-intein (IC). Mixing the two intein-modified Savinase peptide fragments triggers the intein-mediated association of the fragments, splicing the inactive fragments and seamlessly connecting them into a complete, functional enzyme. The first step in developing intein-modified Savinase is to analyze potential intein insertion sites in the Savinase protein sequence. Inteins require the presence of serine (S), threonine (T) or cysteine (C) residues on the C-terminal side of the intein insertion site to allow the splicing reaction to occur. There are no cysteine residues in Savinase, leaving only natural serine and threonine sites for selection without having to mutate the native enzyme sequence. All natural intein insertion sites were computationally analyzed, and more than 20 sites were experimentally tested using either model inteins or trans-spliced inteins of the cis-spliced version (essentially connecting the two halves of the trans-spliced intein via a small peptide bridge). Based on this analysis and experimental data, four initial sites were selected as intein insertion sites: serine 135 (S135), serine 293 (S293), serine 317 (S317), and threonine 318 (T318).

After evaluating the intein insertion sites, five trans-splicing (or "split") inteins were selected for development of regulated trans-splicing. The selected trans-splicing inteins were: NrdJ-1, Gp41-1, IMPDH-1, Gp41-8, and SSPDnaE. These inteins were used to construct trans-splicing iSavinase molecules and expressed in E. coli. Of these inteins, Gp41-1 provided significantly mature and activated trans-splicing iSavinase at insertion site 317 in a detergent inhibition assay as shown in Figure 10. Referring to the figure, pProtein samples were run on SDS-PAGE and stained with Coomassie Brilliant Blue dye to visualize protein bands. In this figure, NI represents the amino-terminal intein-modified peptide of iSavinase (appearing on the gel at approximately 48 kDa), IC represents the carboxyl-terminal intein-modified peptide of iSavinase (appearing on the gel at approximately 12 kDa), NI+IC represents a mixture of the NI and IC peptides, and the host lysate shows background protein bands from the untransformed E. coli host. In these lysates, as well as in the NI+IC mixture, when formulated in a 50% (v/v) detergent concentration, the NI and IC fragments are clearly visible. In contrast, the NI+IC formulation in water shows little or no residual NI and IC bands, respectively, and shows clear bands of other protein degradation products in the lysate, indicating fully active Savinase.

These constructs were further tested in a dilution assay to determine the activity of unmodified Savinase, NI, IC, and a mixture (NI+IC), relative to that observed in a wash application in ~100% detergent and when diluted from 100% detergent to <1% detergent. To ensure the presence of sufficient enzyme to obtain a measurable signal in the dilution assay described below, the protein was concentrated using acetone precipitation or a MW cutoff filter before formulation into detergent. The concentrated protein was then used for formulation and testing in the dilution assay. FIG11 shows a dilution assay using detergent-regulated, trans-splicing iSavinase: S317-Gp41-1 NI and IC. Referring to the figure, equal volumes of iSavinase-NI and iSavinase-IC in mTSB-Ca supplemented with 10 mM DTT were mixed with 50% detergent in water in four different assembly orders. The final concentration of detergent in the mixture was 25%. After incubation overnight at room temperature, aliquots from each sample were diluted into water and tested for detergent and iSavinase activity over 120 minutes using the succinyl FAAF-pNA substrate. All aliquots diluted into water exhibited protease activity, while all aliquots diluted into detergent exhibited no activity. As shown in the figure, the NI+IC formulation was inactive when diluted into detergent; however, significant activity was regained upon dilution into water. NI and IC were inactive regardless of dilution into water or detergent, respectively. In these experiments, a colorless detergent formulation was used, allowing activity to be measured directly in detergent using a standard peptide substrate.

Because these constructs demonstrated significant detergent dilution modulation, more proteins were prepared and used in decontamination assays using test fabric discs. The discs were stained with blood or a combination of blood, milk, and ink. Initial attempts to use NI and IC lysates for decontamination were unsuccessful in demonstrating significant levels of decontamination, suggesting that NI and IC formulations containing high concentrations of salt (a component of commonly used trans-splicing buffers) might inhibit decontamination due to their higher salt concentrations. In fact, when a mixture of NI and IC was used in a decontamination assay at low salt concentrations, significant decontamination effects were observed as shown in Figures 13 and 14.

Figure 12 shows the removal of blood stains using trans-splicing iSavinase. Different volumes of NI+IC lysate were prepared and loaded onto stained fabric discs to examine the effect of concentration on stain removal. Similarly, different concentrations of Savinase Ultra 16L were loaded onto the stained fabric discs. Water, trans-splicing buffer, and mTSB served as negative controls. Referring to this figure, iSavinase was prepared by trans-splicing purified iSav-NI and iSav-IC in mTSB-Ca supplemented with 1 mM DTT at 37°C for 80 minutes, desalted on a Zeba Spin Desalting 7K Mwco column (Thermo Fisher), and then stored on ice. The iSavinase concentration was ~0.94 μg/μL. A control, Savinase Ultra 16L (approximately 103 g/L), was freshly diluted into deionized water at 1:1000, 1:5000, and 1:10,000 (v/v) and stored on ice. Five replicates were performed for each treatment. The following reagents were added to each well of a fabric disc contaminated with dried blood: 20 μL of 10× detergent (2.5% v/v in deionized water), 20 μL of 10× boric acid 200 mM (pH 9.0), and 6 μL of 120FM (8 mM calcium chloride and 4 mM magnesium chloride in deionized water). Water was added to wells 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 in volumes of 149, 144, 134, 104, 54, 129, 134, 134, 134, 154, and 149 μL, respectively. mTSB-Ca supplemented with 1 mM DTT was added to wells 6 and 11 at 5 μL per well. Finally, enzymes were added to bring the total sample volume to 200 μL: iSavinase was added to wells 1, 2, 3, 4, and 5 in increasing volumes of 5, 10, 20, 50, and 100 μL, respectively. Savinase Ultra 16L indicated dilution was added to wells 6, 7, 8, 9, and 10 at a volume of 20 μL per well. The samples were mixed using a pipette, and the plate was incubated at 37°C for 1 hour. The supernatant was removed, and 200 μL of deionized water was added to each well. The plate was then placed on a shaker for approximately 45 seconds. The supernatant was removed, the wash step was repeated two more times, and the plate was dried at ambient temperature overnight. Trans-splicing iSavinase was observed to provide significant decontamination, starting with a 50 μL load of lysate.

Figure 13 shows the use of trans-splicing iSavinase to remove blood, milk, and ink stains. iSavinase was prepared by trans-splicing purified iSav-NI and iSav-IC in mTSB-Ca supplemented with 1 mM DTT at 37°C for 80 minutes, desalted on a Zeba Spin Desalting 7K Mwco column (Thermo Fisher), and stored on ice. The concentration of iSavinase was approximately 0.94 μg/μL. A control, Savinase Ultra 16L (approximately 103 g/L), was freshly diluted in deionized water at 1:100, 1:500, 1:1000, 1:5000, and 1:10,000 (v/v) and stored on ice. Three replicates were performed for each treatment. To each well of a fabric disc contaminated with dried blood, milk, and ink, the following reagents were added: 20 μL of 10× detergent (2.5% v/v in deionized water), 20 μL of 10× boric acid 200 mM (pH 9.0), and 6 μL of 120 FM (8 mM calcium chloride and 4 mM magnesium chloride in deionized water). Water was added to wells 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 in volumes of 149, 144, 134, 104, 54, 134, 134, 134, 134, 154, and 54 μL, respectively. Finally, enzyme was added to a total sample volume of 200 μL: desalted iSavinase was added to wells 1, 2, 3, 4, and 5 in increasing volumes of 5, 10, 20, 50, and 100 μL, respectively. To well 12, 100 μL of iSavinase was added to mTSB-Ca supplemented with 1 mM DTT. Savinase Ultra 16L dilution was added to wells 6, 7, 8, 9, and 10 at a volume of 20 μL per well. The samples were mixed using a pipette, and the plate was incubated at 37°C for 1 hour. The supernatant was removed, and 200 μL of deionized water was added to each well. The plate was then shaken for approximately 45 seconds. The supernatant was removed, and the wash step was repeated two more times. The plate was then dried at ambient temperature overnight.

Different volumes of NI+IC lysate were prepared and loaded onto stained fabric discs to test the effect of concentration on the decontamination effect. Similarly, different concentrations of Savinase Ultra 16L were also loaded onto stained fabric discs. Water was used as a negative control. In order to show the decontamination effect of TSB, 100 μL of NI+IC was prepared with the same 100 μL of TSB. Although iSavinase was fully activated under the two sets of conditions determined by the activity assay, it showed measurable decontamination inhibition (compare lanes 5 and 12). Based on these results, iSavinase trans-splicing starting from a load of 50 μL lysate provided significant decontamination performance. Over time, NI and IC formulations in >90% detergent formulations were tested. In the time period (up to 17 days) test, these formulations showed very significant detergent-dilution-regulated activity retention. In contrast, unmodified Savinase, prepared and formed in the same manner, lost most of its activity within five days. These results indicate that formulating NI and IC in detergents has significant and unexpected benefits relative to unmodified Savinase, which rapidly loses its activity and therefore requires significantly higher enzyme concentrations to ensure activity throughout the useful life of the detergent.

Figure 14 shows the stability testing of trans-splicing iSavinase: S317-Gp41-1 NI and IC detergents. The formulated NI+IC samples were tested over a period of 17 days and showed sustained activity in the detergent dilution test. Referring to the figure, iSavinase-NI and iSavinase-IC lysates were precipitated with acetone, respectively, and formulated into detergents as described in the Materials and Methods. The formulated samples were stored at room temperature, and the stability of the formulated iSavinase-NI and -IC was tested in the detergent dilution test over 17 days. Aliquots diluted into water showed activity at every time point, while aliquots diluted into detergent had no activity at all time points. In contrast, the unmodified Savinase lost a significantly greater portion of its activity when formulated and assayed over the same time period.

Example 22 Dilution-regulated cis-splicing iSavinase

Based on analysis of the unmodified Savinase enzyme, 20 different potential intein insertion sites were experimentally evaluated through activity assays and Western blotting using model intein peptides. To continue the research, the evaluation focused on attempts to target the putative intein insertion sites Serine 135 (S135) and Serine 317 (S317) in Savinase. 60 intein peptides, primarily selected from mesophilic host organisms, were used to screen the S317 site in dilution-induced Savinase. The resulting iSavinase construct was expressed in Escherichia coli, and lysates were tested in detergent inhibition and detergent (dilution) induction assays to measure their performance under these conditions. Figure 15 shows a detergent inhibition test for the cis-splicing iSavinase construct. Equal amounts of total protein from the iSavinase lysate were formulated in detergents at varying concentrations. Individual iSavinases were constructed using different intein peptides in the S317 insertion site. Activity assays were performed on each formulated lysate, and plotted against detergent concentration. Each bar graph represents the mean of 8 biological replicates, and the error bars represent the standard deviation of the measurements. As shown in Figure 15, significant inhibitory activity was observed when the different iSavinase lysates were formulated with detergent concentrations greater than 10%. Among the different inteins tested, the Hwa-MCM1 intein showed significant activity inhibition even when the detergent concentration was below 5%.

Based on the positive results obtained in detergent inhibition, all iSavinase NICs were screened by the detergent dilution induction assay to potentially identify NICs that, despite being initially formed in high detergent concentrations, became active in a low detergent environment.

To maintain appropriate protein concentrations in the final assay, while still reducing detergent concentrations to below 10%, aliquots of NICs were diluted 8-fold or 400-fold into water, and their activities were compared. Figure 16 shows a detergent dilution analysis of cis-spliced iSavinase constructs. Equal amounts of total protein from iSavinase lysates were formulated at a detergent concentration of 25% (v/v). Each iSavinase molecule was diluted 1:8 (open bars) or 1:400 (solid bars) in water, and then assayed for activity. Each bar represents the mean of eight biological replicates, and error bars represent the standard deviation of the measurements. It was observed that the Hwa-MCM1 intein exhibited significant modulation by detergent dilution. iSavinase:Hwa-MCM1 was selected for further study. This molecule underwent a single round of mutagenesis, from which variants with slightly improved regulated activity were selected. In stability testing, the leading candidate molecule did not maintain significant activity after five days, and expression levels were not high enough to demonstrate significant detergent removal performance. In dilution tests, it has elevated background activity relative to the dominant trans-splicing iSavinase molecule. Because the molecule splices in an aqueous environment, the protein tested in the lysate is expected to be a mixture of soluble iSavinase molecules, insoluble iSavinase molecules, and spliced mature Savinase molecules. This mixture is consistent with the elevated background activity observed in the presence of detergent. It is unclear whether the stability of the molecule is an inherent property or is due to the expression, purification, and formulation processes.

Example 23 Cold-regulated cis-splicing iSavinase

Cis-splicing iSavinase molecules are designed to be activated in aqueous environments but inactive at high concentrations in formulated detergents. Because these molecules are likely to undergo splicing and become active during enzyme expression, harvesting, and formulation in aqueous solutions, it is necessary to consider other stimuli that could complement detergent regulation as a means of controlling iSavinase activity. Complementary methods for regulating intein splicing enable efficient preparation and formulation of precursor NIC molecules into detergent products while still providing the desired performance properties. Cold induction was used as a secondary splicing stimulus, which could also be used as an alternative to identify detergent-regulated iSavinase molecules.

Because yeast is highly sensitive to the expression of heterologous proteases, it was chosen as the preferred expression host for screening cold-inducible iSavinase molecules. Experiments using yeast also provided a means of rapidly constructing iSavinase NIC genes by exploiting yeast's inherent ability to direct homologous recombination between qualified, co-transformed DNA fragments. Using this approach, 157 different inteins were inserted into the S135 and S317 sites of previously selected intein-producing clones expressing iSavinase NICs of SEQ ID NOs: 140-453 and 496. Of particular interest were intein constructs of SEQ ID NOs: 634-671, which encode proteins having the amino acid sequences of SEQ ID NOs: 497-535.

Once constructed, recombinant yeast clones were used for growth inhibition screening. Yeast clones expressing iSavinase NICs were compared to recombinant yeast strains expressing unmodified Savinase and a recombinant yeast strain expressing a mutant, inactive iSavinase enzyme (referred to herein as H62A; SEQ ID NO: 682) at 20°C and 30°C. Clones that grew well at 30°C, comparable to the H62A yeast strain expressing the inactive Savinase, and clones that grew poorly at 20°C, comparable to the yeast strain expressing unmodified (native) Savinase, were selected for further evaluation. To perform the screening correctly, eight colonies were selected for each of the 314 constructs SEQ ID NOs: 140-453 and 496 (the 157 intein was inserted at two different sites in Savinase) and cultured overnight in Ura- and glucose+ media, without inducing iSavinase gene expression. These cultures were used to inoculate expansion cultures at the same OD value and grown at 20°C and 30°C in Ura- and glucose-containing media under conditions that regulated iSavinase gene expression. The cultures were then monitored over 72 hours, and constructs that reproducibly demonstrated significant growth differences relative to the control at each temperature were selected for validation of their regulated Savinase activity. Growth differential validation was performed using a temperature-induced assay. In the assay, iSavinase NICs were prepared at either 30°C (yeast) or 37°C (E. coli; this host was used to further validate performance in yeast), harvested, and divided into two aliquots. One aliquot was incubated at 20°C for two hours, while the other aliquot was maintained at its preparation temperature (30°C or 37°C for two hours). After the two-hour incubation, the 20°C incubated sample was warmed to its preparation temperature. Samples were removed from both aliquots, mixed with substrate, and assayed at the preparation temperature to compare the uninduced and induced activities of the iSavinase molecules. Figure 17 shows the results of temperature-induced validation assays of selected cis-splicing iSavinase constructs: iproSavinaseS135:15 (Chth_ATPase_BIL; SEQ ID NO: 154, 545), iproSavinaseS135:145 (Tko_Pol-2_Pko_pol-2; SEQ ID NO: 284, 553), iproSavinaseS135:153 (Tvo_VMA; SEQ ID NO: 292, 555), iproSavinaseS135:155 (UNC-ERS_RIR1; SEQ ID NO: 294), iproSavinaseS135:155-var7 (improved mutant; SEQ ID NO: 496, 633), as well as control constructs ProSavinase (SEQ ID NO: 57) and proSaviH62 (SEQ ID NO: 682, 683). Selected iSavinase NICs were mutated and screened in two consecutive rounds of assays to further improve their performance. Referring to the figure, cold-induced Savinase activity was tested using in vitro prepared proteins. Shown here are representative examples of intein-modified proSavinases from a screened proSavinase library containing native inteins (iproSavinaseS135:15, SEQ ID NO:545; iproSavinaseS135:145, SEQ ID NO:553; and iproSavinaseS135:153, SEQ ID NO:554) and a mutagenized mutant (proSaviS317:155var7, SEQ ID NO:633). The parent from which proSaviS317:Unc-ERS_RIR1-var7 was derived was proSaviS317:Unc-ERS_RIR1. ProSavinase (unmodified Savinase) and proSaviH62A (inactive Savinase) were used as controls. Reactions without DNA template also served as controls. Savinase activity differed significantly between 20°C (open bars) and 37°C (solid bars), with enhanced activity for all constructs at 20°C.

Figures 18A-18C show the time course of Savinase activity for two leading cold-induced iSavinases: iproSavinaseS135:Cth_ATPase-BIL (SEQ ID NO: 154, 545) (Figure 18B) and iproSavinaseS135:Mja_Klba (SEQ ID NO: 344) (Figure 18C), compared to unmodified proSavinase (SEQ ID NO: 57) and inactive proSavinaseH62A (SEQ ID NO: 682, 683) (Figure 18A). Proteins from unmodified proSavinase, an inactive Savinase (proSavH62A), and intein-modified proSavinases (proSaviS135:Cth_ATPase_BIL and proSaviS135:Mja_Kiba) were treated at 20°C and 37°C for 2 hours to induce intein splicing, followed by incubation with N-succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroanilide for 1 hour at 37°C. Absorbance (415 nm) was measured every 2 minutes for 30 minutes. As shown in Figures 18B and 18C, the activity of two iproSavinase clones was induced by low-temperature treatment (20°C), but not in the controls (proSav and SavH62A).

Selected cold-regulated iSavinase NICs were also tested for detergent-dependent regulation using detergent inhibition and induction assays at 20°C or 37°C. Figures 19A-19F show the time course of activity of proSavinaseS135:Cth_ATPase_BIL (SEQ ID NOs: 154, 545) and a control construct, ProSavinase (SEQ ID NO: 57), as well as the inactive proSaviH62 (SEQ ID NOs: 682, 683), in detergent formulations. Due to the difficulty in expressing certain NIC molecules to significant levels in yeast or E. coli systems, in vitro transcription and translation (IVTT) methods were used to prepare NICs for detergent-dependent regulation testing. Proteins from unmodified proSavinase ( Figures 19A and 19B ), inactive Savinase (SavH62A; Figures 19C and 19D ), and intein-modified proSavinase (proSaviS135:Cth_ATPase_BIL; Figures 19E and 19F ) were mixed with various detergent formulations to produce final concentrations of 25%, 5%, 1%, and 0.2% detergent and then treated at 20°C or 37°C for 2 hours to induce intein splicing. The cells were then incubated with N-succinyl-alanyl-alanyl-prolyl-phenylalanine p-nitroanilide substrate at 37°C. Absorbance (405 nm) was measured every 2 minutes for 90 minutes. The activity of proSavinase S135:Cth_ATPase_BIL varied in a detergent concentration-dependent manner, with no activity in a high-concentration (25%) detergent formulation and high activity in a low-concentration (0.2%) detergent formulation. Activity was also induced by a 2-hour treatment at 20°C. However, the activity of the protease in controls (proSavinase and proSavinase H62A) remained unchanged at varying detergent concentrations. Inhibition of intein splicing was also observed in high-concentration (greater than or equal to 25%) detergent formulations. This behavior is consistent with the temperature-regulated protease activity previously established for this molecule ( Figure 17 ).

Figures 20A-20D show the time course of Savinase activity of cis-spliced iSavinase constructs iproSavinaseS135:Cth_ATPase_BIL (S135:15 as shown in Figure 20C), iproSavinaseS135:Mja_Klba (S135:48 as shown in Figure 20C) in a cold-induced detergent dilution induction assay at 20°C and 37°C, compared to unmodified proSavinase (Figure 20A) and inactive proSavinase H62A (Figure 20B). Equal amounts of protein from unmodified proSavinase, inactive Savinase (proSavinase H62A), and intein-modified proSavinase (proSaviS135:Cth_ATPase_BIL and proSaviS135:Mja_Kiba) were mixed with a 37°C detergent formulation to a final concentration of 25%. The samples were then diluted with water or BR buffer (pH 9.0) at either 20°C or 37°C and maintained in this condition for 2 hours to induce intein splicing. The samples were then incubated with the substrate N-succinyl-alanyl-alanyl-prolyl-phenylalanine p-nitroanilide at 37°C. Absorbance (415 nm) was measured every 2 minutes for 90 minutes. Savinase activity increased when diluted in water or BR buffer at 20°C but remained unchanged at 37°C. This trend was observed only in intein-modified proSavinase (S135:15 in Figure 20C and S135:48 in Figure 20D), but not in controls (proSavinase in Figure 20A and SavH62A in Figure 20B). This suggests that upon dilution with water, intein splicing is relieved from high-concentration detergent inhibition. In Figure 20C, S135:15 represents the proSavinaseS135:Cth_ATPase_BIL construct. In Figure 20D, S135:48 represents the proSavinaseS135:Mja_Kiba construct. Of the molecules tested, only iproSavinaseS135:48 showed some degree of modulation by detergent dilution. All iSavinase NICs molecules were observed to exhibit significant cold-temperature modulation and were strongly inhibited by high-concentration detergent formulations.

Example 23 Method Development

Protease screening assays and various analytical methods have been developed to test enzyme activity, and several different expression systems have been developed for enzyme screening and production.

A method for screening engineered iSavinase molecules was developed using agar plates containing a colorimetric substrate that indicates the presence of active protease upon cleavage. These plates were initially used in the screening process to rapidly evaluate constructed NICs based on their activity at different temperatures by counting microbial colonies. Because this assay is not quantitative, it enables rapid screening of libraries.

Quantitative assays using soluble substrates have also been developed that can measure both pre- and post-splicing activity. These assays have been automated in 96- and 384-well plates. In one assay for these proteases, the substrate Suc-Phe-Ala-Ala-Phe-4NA is used, which is known to have a _kcat / _Km value 10 times greater than that of the alkaline protease Suc-Ala-Ala-Pro-Phe-4NA commonly used in subtilisin. This more specific substrate has been experimentally determined to provide the strongest signal-to-noise ratio in control experiments and makes automated screening for Savinase more feasible. Both substrates can be used to directly measure protease activity in clear, colorless detergent formulations. These formulations are important for both screening molecules and directly measuring activity in formulations. In addition, epitope-specific and polyclonal antibodies are used against unmodified proSavinase used in Western analysis.

Initially, Escherichia coli and Bacillus subtilis expression systems were developed. The E. coli system is used for cytoplasmic or periplasmic expression, while the B. subtilis-based system is used for protein secretion. While the Bacillus system has a relatively low throughput, it may be useful in the future for preparing candidate enzymes. High-throughput screening systems were developed using E. coli. Unmodified Savinase, proSavinase, and intein-modified forms of the enzyme were expressed using these systems. In addition, yeast-based expression systems were also developed. Finally, the results showed that IVTT could be effectively used in the preparation of iSavinase and unmodified Savinase molecules. In summary, these different expression systems provide screening capabilities and protein preparation capabilities to obtain intein-modified Savinase that meets detergent application specifications.

iSavinase-NI and iSavinase-IC expression. Trans-splicing iSavinase pairs iSav-NI and iSav-IC were individually expressed in E. coli. Expression was driven by a T7 promoter and induced by IPTG in the pET-Duet1 vector and the E. coli BL21-Gold (DE3) strain. Unmodified proSavinase was used as a positive control, and empty vector was used as a negative control.

Overnight cultures were grown in LB medium supplemented with 100 mg/L ampicillin. Aliquots were inoculated into fresh medium and grown to an _OD600 of 0.6 before adding IPTG. After 3 hours, cells were harvested by centrifugation.

The trans-splicing constructs in E. coli stored in glycerol are as follows: pETDuet1-iSav-NI-GG-6His (iSav-NI) in BL21Gold(DE3); pETDuet1-iSav-IC-Sumo-6His (iSav-IC) in BL21Gold(DE3); pETDuet1-proSavinase (positive control) in BL21Gold(DE3) and pETDuet1 (negative control) in BL21Gold(DE3). The trans-splicing constructs in E. coli were quickly transferred from the glycerol stock to LB+carb 100 mg/L agar plates and incubated at 30°C overnight. The plates were removed from 30°C and used immediately to inoculate liquid cultures or stored at 4°C for up to 1 week. 4 mL of LB carb 100 mg/L medium was aliquoted into four 17×100 mm polystyrene tubes or similar. The single bacterium colony from the agar plate is inoculated into the starting culture of 4mL, and incubated overnight at 30°C, 300rpm. Each 2.5mL overnight starting culture is inoculated into 100mL fresh culture medium (40 × dilution), and incubated at 30°C, on a shaking table with 300rpm, until OD ₆₀₀ reaches 0.6. It is 0.5mM to add IPTG to a final concentration. The culture is further incubated for 3 hours at 30°C, 300rpm. Take a 30mL aliquot and granulate the cells at 15°C for 10 minutes at 3000g. Test tube is placed on ice, and the supernatant is discarded. As described in the "Harvest of iSavinase-NI, iSavinase-IC" part, start the preparation of protein immediately, or at -80°C, the cell pellet is stored in a 50mL Falcon tube.

Preparation of mTSB+Ca buffer. A modified trans-splicing buffer (TSB) contains 50 mM Tris base (Trizma ^™ ), 150 mM sodium chloride, 2 mM calcium chloride, and 1 mM DTT (dithiothreitol). TSB containing 2 mM calcium (mTSB+Ca) is used for trans-splicing enzyme assays in cell lysis buffer and for dialysis of cell lysates solubilized with iSavinase NI and IC.

Harvesting of iSavinase-NI and iSavinase-IC. Trans-spliced proteins (iSavinase-NI and -IC) expressed in E. coli were harvested. iSavinase-NI was partially soluble, while -IC was nearly insoluble. To solubilize the trans-spliced proteins, cell lysates were dissolved in 6 M urea. Urea was removed by overnight dialysis, and the soluble protein fraction was collected.

800 μL of lysis buffer (containing 400 μL of 10× Fastbreak Cell Lysis Reagent, #2013-10-13 (Promega) (10×), 3600 μL of mTSB+Ca, and 8 μL of Benzonase (Nuclease HC, #71205-3, Novagen) was added to a 30 mL cell pellet in a 50 mL conical Falcon tube, vortexed and pipetted until the pellet was completely suspended. The cells were placed at room temperature for 25 minutes. 1500 μL of 10 M urea solution was added to mTSB+Ca at a ratio of lysate to urea of 4:6 (v/v) and incubated at 300 rpm at 25°C for 120 minutes. Each urea-solubilized lysate was transferred to two 1.5 mL microtubes. The lysate was clarified at 5000 g for 5 minutes. The supernatant containing the urea-solubilized protein fraction was transferred to a Tube-O-Dialyzer Medi 4 kDa The cells were placed in MWCO tubing (17×100 mm polystyrene tubing #1485-2810; USA Scientific) and dialyzed against mTSB+Ca at room temperature overnight.

The dialysis buffer was replaced and dialysis was continued at room temperature for 2 hours. The dialysis tubing was gently vortexed with the membrane facing downward to remove proteins from the membrane. The sample was transferred from the Tube-O-Dialyzer to two 1.5 mL microtubes and spun at 5000 g for 5 minutes. The supernatant was collected into a 15 mL acetone-resistant conical polypropylene tube (Falcon). The sample was kept at room temperature. The solubilized iSav-NI and iSav-IC lysates were further tested for trans-splicing activity.

Detergent formulation of purified iSav-NI and iSav-IC. Purified iSavinase-NI (MW 42 kDa) and iSavinase-IC (MW 27 kDa) were redissolved in mTSB-Ca + 10 mM DTT and dissolved in equimolar amounts of detergent. The mixture was mixed at a molar mass ratio of NI:IC = 1.6:1. Briefly, a 50% (v/v) solution of the detergent MTS24 (Maradona 10) was prepared in water. iSavinase-NI (3.8 mg/mL in 150 mM NaCl, 50 mM MES, pH 6.3, and 40% glycerol) and -IC (2.59 mg/mL in 10 mM Tris, pH 8.0, and 40% glycerol) were redissolved in mTSB-Ca + 10 mM DTT. In a PCR tube, 22 μL (~84 μg) of iSavinase-NI and 22 μL of mTSB-Ca + 10 mM DTT were pipetted together. The mixture was briefly spun to remove air bubbles. In a separate PCT tube, 22 μL (~57 μg) of iSavinase-IC was pipetted together with 22 μL of mTSB-Ca + 10 mM DTT and spun briefly to remove air bubbles. The mixture was incubated at room temperature for 30 minutes. iSavinase-NI and -IC were formulated in detergent in four different assembly orders. In the PCR tube, the formulations were formed after thorough mixing and waiting approximately 1 minute after adding each component. The final concentration of detergent was adjusted to 25%. The formulation samples were: 10 μL of 50% detergent + 5 μL of NI + 5 μL of IC; 10 μL of 50% detergent + 5 μL of IC + 5 μL of NI; 5 μL of IC + 10 μL of 50% detergent + 5 μL of NI; 5 μL of NI + 10 μL of 50% detergent + 5 μL of IC; 10 μL of 50% detergent + 5 μL of NI + 5 μL of mTSB (control); 10 μL of 50% detergent + 5 μL of IC + 5 μL of mTSB (control). The tubes were briefly vortexed and incubated overnight at room temperature to promote assembly of the trans-splicing pair. The detergent dilution assay was run on a 5 μL aliquot (see Detergent Dilution Analysis of Purified Formulated Trans-Splicing iSavinase).

Inhibition and activation of trans-splicing in water and savinase by detergent. Trans-splicing activity was evaluated in water and detergent. In water, trans-splicing restored savinase activity. In detergent, trans-splicing was inhibited, and no protease activity was detected.

Purified iSavinase-NI in 150 mM NaCl, 50 mM MES, pH 6.3, and 40% glycerol included the following samples: 250 μL of iSavinase-NI (2) (2.6 mg/mL; monomer); 300 μL of iSavinase-NI (3) (3.8 mg/mL; monomer); 800 μL of iSavinase-NI (4) (8.6 mg/mL; dimer); and 800 μL of iSavinase-NI (5) (7.1 mg/mL).

Purified iSavinase-IC in 10 mM Tris, 40% glycerol, pH 8.0 contained 11 mL of iSavinase-IC (2.59 mg/mL). pET Duet1 proSavinase was used as a positive control, and pET-Duet1 empty vector as a negative control.

3× purified protein samples were prepared in pre-labeled PCR tubes as follows: NI2 (16 μL of iSav NI-2 sample in 14 μL of mTSB+Ca+DTT buffer); NI3 (11 μL of iSav NI-3 in 19 μL of mTSB+Ca+DTT buffer); NI4 (5 μL of iSav NI-4 in 5 μL of iSav NI-4 buffer); NI5 (6 μL of iSav-NI-5 in 24 μL of mTSB+Ca+DTT buffer); IC (10 μL of iSav-IC in 20 μL of mTSB+Ca+DTT buffer); “-” (10 μL of pET-DUET in 20 μL of mTSB+Ca+DTT buffer) and “+” (10 μL of Savinase in 20 μL of mTSB+Ca+DTT buffer). Samples were kept at room temperature for 30 minutes. Paper well templates are used to organize sample placement before starting the assay.

Two conditions were tested.

(1) Test activated in water ( _H2O +NI+IC): _dH2O was pre-filled in a PCR tube, and the previously collected 3× purified protein was added to a final concentration of 1×. A 30 μL sample included 24.0 μL of _dH2O and 3 μL of 3× protein 1 and 3× protein 2, respectively.

(2) Detergent inhibition test (100% detergent + NI + IC): 100% detergent was pre-filled into a PCR tube, and the previously collected 3× purified protein was added to a final concentration of 1×. 30 μL of sample included 24.0 μL of 100% detergent and 3 μL of 3× protein 1 and 3× protein 2, respectively. The sample was incubated at 37°C for 30 minutes and then loaded onto a Savinase test plate, which was a 96-well flat-bottom plate (Costar 9017: flat bottom, medium binding). For the test activated in water, the sample was transferred to 6 wells pre-filled with 60 μL of H ₂ O per well. For the detergent inhibition test, the sample was transferred to 6 wells pre-filled with 60 μL of 100% detergent per well. 10 μL of 1× BR buffer at pH 9.0 and 100 μL of 2× substrate (500 μM succinyl-FAAF-pNA in 20% DMSO) were added to each well. The spectrophotometer was set to a kinetic reading. The activity of the sample was measured kinetically by recording the absorbance at 400 nm once every minute for two hours.

Detergent inhibition of trans-splicing in cell lysates

Detergent inhibition of cell lysates using iSavinase-NI and -IC was evaluated. Cell lysates of iSavinase-NI and -IC were mixed, detergent was added, and the samples were used for the Savinase activity assay. Briefly, 60 μL of 100% detergent was preloaded into 5 wells of a 96-well flat-bottom plate. The following cell lysates were added: 15 μL of NI + 15 μL negative control (empty vector cell lysate); 15 μL of IC + 15 μL negative control; 15 μL of NI + 15 μL of IC; 30 μL negative control and 15 μL positive control (Savinase) + 15 μL negative control (empty vector lysate). The plate was incubated at 37°C for 1 hour. 10 μL of 1× BR buffer, pH 9.0, and 100 μL of 2× substrate (500 μM succinyl-FAAF-pNA in 20% DMSO) were added to each well. The activity of the samples was measured kinetically by recording the absorbance at 400 nm once every minute for two hours.

Savinase activity was restored from the purified inactive fraction by trans-splicing. The restoration of Savinase activity by trans-splicing of iSavinase-NI (MW.42kDa) and -IC (MW.27kDa) was confirmed. Savinase was split into N- and C-terminal parts, and the trans-spliced intein was attached to the two parts to obtain iSavinase-NI and -IC. Both iSavinase-NI and -IC lacked protease activity. Mixing the two inactive parts triggered trans-splicing, and seamlessly connecting the N- and C-terminal parts of Savinase restored the activity of the enzyme. For efficient trans-splicing, equimolar amounts of NI and NC were mixed at a mass ratio of NI:IC = 1.6:1.

Purified iSavinase-NI in 150 mM NaCl, 50 mM MES, pH 6.3, and 40% glycerol included the following samples: 250 μL of iSavinase-NI (2) (2.6 mg/mL; monomer); 300 μL of iSavinase-NI (3) (3.8 mg/mL; monomer); 800 μL of iSavinase-NI (4) (8.6 mg/mL; dimer); and 800 μL of iSavinase-NI (5) (7.1 mg/mL).

Purified iSavinase-IC in 10 mM Tris pH 8.0 and 40% glycerol contained 11 mL of iSavinase-IC (2.59 mg/mL). pET Duet1 proSavinase was used as a positive control, and pET Duet1 empty vector was used as a negative control.

Briefly, purified protein and mTSB+Ca+DTT were combined in sequentially labeled pre-labeled PCR tubes (1-11) as follows: (1) 1.0 μL of iSav-IC in 29.0 μL of mTSB+Ca+DTT; (2) 1.6 μL of iSav-IC in 28.4 μL of mTSB+Ca+DTT. NI-2; (3) 1.1 μL of iSav-NI-3 in 28.9 μL of mTSB+Ca+DTT; (4) 0.5 μL of iSav-NI-4 in 29.5 μL of mTSB+Ca+DTT; (5) 0.6 μL of iSav-NI-5 in 29.4 μL of mTSB+Ca+DTT; (6) 1.0 μL of iSav-IC and 1.6 μL of iSav-NI-2 in 27.4 μL of mTSB+Ca+DTT; (7) 1.0 μL of iSav-IC and 1.6 μL of iSav-NI-2 in 27.9 μL of mTSB+Ca+DTT. 1 μL of iSav-NI-3; (8) 1.0 μL of iSav-IC and 0.5 μL of iSav-NI-4 in 28.5 μL of mTSB+Ca+DTT; (9) 1.0 μL of iSav-IC and 0.6 μL of iSav-NI-5 in 28.4 μL of mTSB+Ca+DTT; (10) 2.0 μL of pET-DUET in 28.0 μL of mTSB+Ca+DTT; and (11) 1.0 μL of pET-DUET and 1.0 μL of Savinase in 29.0 μL of mTSB+Ca+DTT. The samples were incubated at 37°C for 30 minutes. A Savinase assay plate was prepared with 11 wells pre-loaded with 70 μL of 1× BR buffer, pH 9.0. The samples were transferred to the Savinase assay plate. 100 μL of 2× substrate (500 μM succinyl-FAAF-pNA in 20% DMSO) was added to each well. The activity of the samples was measured kinetically by recording the absorbance at 400 nm once every minute for two hours.

iSavinase-NI and iSavinase-IC lysate preparation. Trans-spliced iSavinase-NI and iSavinase-IC were concentrated and co-solubilized with detergent. iSavinase-NI and -IC were precipitated with acetone, and the acetone suspensions of NI and IC were combined, pelleted, and dissolved in detergent. It is important to verify the trans-splicing activity of the iSavinase-NI and -IC lysates before formulation. The starting material was the clarified urea-solubilized lysate in mTSB-Ca. See Harvest of iSav-NI, iSav-IC. Samples were as follows: iSavinase-NI; iSavinase-IC; proSavinase (positive control), and pET Duet1 empty vector (negative control).

Cell lysate prepared from 30 mL of induced culture cell pellets was dissolved in freshly prepared urea, refolded, clarified and used as starting material (refer to the harvest of iSavinase-NI and iSavinase-IC). The samples were as follows: iSavinase-NI; iSavinase-IC; proSavinase (positive control) and pET Duet1 empty vector (negative control). The volume of the lysate was measured by pipette, and 1 volume of cell lysate was added to 4 volumes of -80°C acetone in a 1 mL aliquot to form a precipitate. The sample was incubated at -80°C for 1 hour. The tube was placed at room temperature to warm for 10 minutes to allow the precipitate to settle to the bottom. The contents of each tube were divided into three equal volumes and placed in 1.5 mL microtubes. Each microtube contained protein from 10 mL of induced culture and was prepared separately. The acetone precipitates of the four samples were divided into 12 microtubes. The contents of one microtube with iSav-NI were added to one microtube with iSav-IC and vortexed gently to mix.

The process was repeated with a second set of iSav-NI and -IC. The samples in the second set were as follows: iSavinase NI+IC (trans-splicing mixture); iSavinase NI (control); iSavinase-IC (control); proSavinase (positive control); and pET Duet1 empty vector (negative control). The samples were vortexed at 13,000 rpm for 10 minutes at room temperature. Acetone was added and evaporated. 4,000 ng of detergent was added to one side of the tube and the contents were mixed by pipette. The samples were incubated at room temperature overnight to continue dissolving the pellet and stored at room temperature. Detergent dilution analysis of cell lysates of formulated trans-splicing iSavinase. The activity of formulated trans-splicing iSavinaseNI+IC cell lysates was evaluated in water and detergent.

Two sets of plates were labeled for water dilution and detergent dilution. Each set had 4 wells for NI+IC, as well as 1 positive control well (Savinase) and a negative control well (pET-DUET empty vector). The NI+IC wells were pre-loaded with 80, 85, 87.5, and 88.75 μL of water or detergent. The control wells were pre-loaded with 88 μL of water or detergent. 10, 5, 2.5, or 1.25 μL of prepared iSavinase NI+IC was added to the NI+IC wells to a final sample volume of 90 TwioL. 2 μL of prepared Savinase was added to the positive control wells. 2 μL of prepared empty vector lysate was added to the negative control wells. 10 μL of 1× BR buffer, pH 9.0, and 100 μL of 2× substrate (500 μM succinyl-FAAF-pNA in 20% DMSO) were added to each well. The activity of the samples was measured kinetically by recording the absorbance at 400 nm once every minute for two hours.

Trans-splicing analysis of iSavinase-NI and -IC in cell lysates

The trans-splicing activity of iSavinase-NI and -IC was evaluated using dissolved clarified cell lysates. The following cell lysates were used: iSavinase-NI and -IC clarified cell lysates, as well as cell lysates as positive controls (pET-Duet1 proSavinase) and negative controls (pET-Duet1 empty vector). The following cell lysates were combined in a flat-bottom plate: 15 μL of NI + 15 μL empty vector; 15 μL of IC + 15 μL empty vector; 15 μL of NI + 15 μL of IC; 30 μL empty vector and 15 μL of Savinase + 15 μL empty vector. Cover the plate with a foil seal and incubate at 37°C for 1 hour. 70 μL of 1× BR buffer, pH 9.0, was added to each sample to a volume of 100 μL. 100 μL of 2× substrate was added to each well. The activity of the samples was measured dynamically by recording the absorbance at 400 nm once every minute for two hours.

Expression of cis-splicing iSavinase in E. coli. Cis-splicing intein-modified iSavinase SS317:31:Hwa and control constructs were expressed in E. coli. To reduce toxicity, expression was targeted to the periplasmic space. E. coli BL21 (DE3) cells containing pET22 constructs of cis-splicing Savinases were distributed as follows: pET22 empty vector control; proSavinase; iproSavinase S317:mTthEU59; iproSavinase S317:31:Hwa_MCM-1; iproSavinase S317:31:HwaA (intein control with no splicing activity); iproSavinase S317:31:Hwa_var (35.G21); and iproSavinase S317:31:Hwa_var (22.C3). Glycerol stocks were stored at -80°C.

From the glycerol stock, clones were quickly moved to agar plates (LB supplemented with 0.5% glucose and 100 mg/L carbenicillin) and single colonies were inoculated into 6 mL of liquid Overnight Express ^™ Instant TB medium, also known as "auto-induction medium" (AIM) (Novagen, EMD Millipore) and incubated at 20° C., 300 rpm for 48 hours. The cells were pelleted at 4000 g for 10 minutes and the supernatant discarded.

E. coli cell lysates were formulated in detergent to demonstrate detergent inhibition of Savinase activity and recovery of activity upon dilution with water. Briefly, 100 μL aliquots of lysate were dispensed into round-bottom 96-well plates. 100 μL of 50, 20, 10, 4, or 2% (v/v) detergent solutions in 1H 9.0 (1× CHES-citrate-HEPES) buffer were added and incubated at 20°C for 2 hours.

Lysates were prepared for determination of Savinase activity in detergent or after dilution in water.

The lysate plate was removed from 20°C and 25 μL of the detergent lysate was transferred to a plate pre-filled with 175 μL of CCH buffer, pH 9.0, or 175 μL of a 25, 10, 5, 2, or 1% detergent formulation in CCH buffer, pH 9.0. The samples were mixed using a pipette. The plate reader was set at 37°C and the absorbance at 400 nm was measured. The enzyme assay was performed as follows. 100 μL of 500 μM FAAF-pNA (N-succinyl-FAAF-pNA; Bachem #L1675; MW 674.71) substrate stock solution was added to a flat-bottom 96-well plate and mixed. 100 μL of substrate was diluted in detergent or CCH buffer, pH 9.0, by pipetting. The absorbance was read at 400 nm over 15-20 minutes.

Determination of Cold-Induced Savinase Activity. PCR reaction components were prepared in the following order: per 50 μL reaction, 33.5 μL nuclease-free water, 10 μL 5× Phusion HF buffer, 1 μL 10 mM dNTPs, 2 μL 10 μM forward primer, 2 μL 10 μM reverse primer, 1 μL template DNA, and 0.5 μL Phusion Hot Start DNA Polymerase (ThermoScientific, cat# F-540L). All components were mixed and briefly centrifuged before use. A miniprep DNA construct carrying ProSavinase NICs (200 ng/μL) was used as the PCR template. Thermal cycling conditions included a 30-second initial denaturation at 98°C, followed by 28 cycles of 98°C for 10 seconds, 65°C for 25 seconds, 72°C for 1.5 minutes, and 72°C for 5 minutes, with a hold at 4°C.

PCR products were purified using the QIAquick PCR purification kit (qiagen). 5 volumes of PB buffer were added to 1 volume of product and mixed, applied to a QIAquick column and centrifuged for 30-60 seconds. The flow-through was discarded, and the QIAquick column was washed with 750 μL of wash buffer PE and centrifuged for 30-60 seconds. To elute the DNA, 50 μL of elution buffer was added to the center of the QIAquick membrane, the column was allowed to stand for 1 minute, and then centrifuged for 1 minute. To assess the concentration of the DNA, 2 μL of the purified PCR product was electrophoresed on a 1% agarose gel with a 2 μL 2-log DNA gradient.

Protein synthesis was performed using the PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, catalog #E6800S). All reagents were kept on ice throughout the reaction. Solutions A and B were thawed on ice and pulse-spinned in a microcentrifuge. 20 μL of the reaction mixture was assembled on ice in a new PCR tube in the following order: 8 μL of solution A, 6 μL of solution B, 6-X μL of _{H 2} O, and X μL of DNA template. The reaction components were gently mixed and pulse-spinned in a microcentrifuge. The mixture was incubated at 30° C. for 2 hours and terminated by mixing with 60 μL of 1× BR buffer, pH 9.0 (preheated to 30° C.). 40 μL of the protein mixture was transferred to a new PCR tube.

To mobilize inteins, 40 μL of the protein mixture was incubated at 20°C or 37°C for 2 hours, followed by an additional 10 minutes at 37°C. 40 μL of 2× substrate stock (pNA substrate; Bachem, cat# L1675) preheated to 37°C was mixed with the sample. After incubation at 37°C for 1 hour, the absorbance was measured at 400 nm.

Screening for Cold-Inducible Savinases in Yeast

Savinases have been shown to cause cytotoxicity when expressed in Escherichia coli and yeast. This selective feature was exploited to develop a cold-inducible intein-modified Savinase. In addition to its high sensitivity to Savinase toxicity, yeast can grow at lower temperatures, making it ideal for cold-induced intein splicing, and exhibits high fidelity/efficiency of homologous recombination, allowing for high-throughput library screening.

Yeast transformation

The yeast expression vector pSavi-Y135/317 was constructed by inserting pro-Savinase into the p416GALL vector (SEQ ID NO: 630) and introducing BamHI recognition sequences at the S135 and S317 sites of Savinase. The pro-Savinase gene was constructed by gap repair and cloning downstream of the GalL promoter of p416GALL. Expression was initiated by galactose and terminated by glucose. pSaviY135 carries the pro-Savinase gene with a BamHI recognition sequence at the S135 site (SEQ ID NO: 631), while pSaviY317 carries the pro-Savinase gene with a BamHI recognition sequence at the S137 site (SEQ ID NO: SEQ ID NO: 632). Vector DNA was typically prepared from an overnight culture of Escherichia coli grown in LB (Luria-Bertani) medium containing ampicillin using the QIAprep Spin Miniprep kit protocol (Qiagen).

To construct the library, BamHI-linearized vector DNA was co-transformed with PCR-amplified intein DNA (Unc-ERS_RIR1 and Sce_VMA), and the transformants were plated on synthetic medium lacking uracil (Ura-) but with glucose or galactose. Yeast strain BY4741 was used to demonstrate a growth inhibitory (cytotoxic) phenotype by heterologous expression of a pro-Savinase gene, which was developed for high-throughput screening assays of Savinase activity generated by intein splicing.

Conventional yeast transformation was performed using the LiAc/SS carrier DNA/PEG method. 2 μL of BamHI-linearized pSaviY135/317 and 6 μg of PCR-generated intein variants were mixed with 400 μL of freshly prepared yeast competent cells and placed in a GenePulser tube (0.2 cm gap) for transformation at 2.5 kV and 25 μF (typical time constants ranged from 3.0 to 4.5 milliseconds). This electroporation method allows for the efficient generation of large libraries with up to 4×10 ⁷ variants.

After electroporation, the yeast transformation mixture is plated on -Ura agar plates containing 2% galactose (which is driven by the GalL promoter) and cultured at 30°C for 3 days. Yeast cells carrying variants that constitutively splice at 30°C will accumulate active Savinase, leading to growth inhibition or elimination of the host cells. This results in a sub-library enriched for yeast transformants whose splicing is inhibited at 30°C. This process typically produces an enrichment of approximately 100-fold.

Savinase activity-dependent yeast growth inhibition was developed in a cell-based selection assay, which was used in a primary library screen to identify cold-inducible iSavinases. Following library enrichment on 2% galactose, yeast transformants were individually picked as colonies and inoculated into 0.5 or 0.1 mL of selective medium containing 2% glucose and without Ura. After incubation at 30°C for 2 days, saturated yeast cultures ( _OD600 of 3-4) were subcultured (100-fold dilution) in selective medium containing 2% galactose and incubated in two sets of 96- or 384-well plates, one at 20°C for 5 days and the other at 30°C for 5 days. Cell growth was monitored daily by measuring _OD600 . Unmodified pro-Savinase and its mutant H62ASavinase construct served as controls throughout the assay. Cells expressing unmodified pro-Savinase grew poorly at 20°C and 30°C, while cells expressing the inactive H62A Savinase grew well at both temperatures. Yeast mutants that grew normally at 30°C, similar to H62A-expressing cells, but grew very slowly at 20°C, similar to unmodified pro-Savinase, were scored as "positive." For validation, positive clones were then cherry-picked and re-evaluated for growth phenotypes at 20°C and 37°C. After validation, 54 clones of the Unc-ERS_RIR1 mutant and 60 clones of the Sce_VMA mutant were prioritized as "HITs" for further evaluation and as lead candidates in secondary (activity) assays. Vector DNA was prepared and used for sequence analysis of the mutated intein mutants.

Screening of natural inteins in Savinase

157 inteins were PCR amplified and inserted into the S135 and S317 sites via yeast homologous recombination. Equimolar amounts of BamHI-linearized pSavi-Y and the PCR-amplified inteins were mixed with SS-DNA, PEG, and LiCl, along with competent yeast cells. After incubation at 30°C for 30 minutes, the mixture was heat-shocked at 42°C for 15 minutes. The cell pellet was resuspended in _H₂O and plated on selective agar plates containing galactose at 30°C for two days. Eight colonies were grown to saturation on non-selective glucose liquid medium (30°C, 2 days), and small aliquots (2.5 μL) were inoculated into replicates of selective galactose medium. One set was cultured at 30°C and the other at 20°C without shaking before OD _590nm measurements were obtained for all samples. Clones that grew well at 30°C but very slowly at 20°C were selected for further testing.

Screening of mutagenized inteins in Savinase

Several inteins (Kra_DnaB, Pho_IF2, Pho_r-Gyr, Unc-ERS_RIR1, and SceVMA) were mutagenized by PCR to insert Savinase at the S317 site. The sceVMA intein was also inserted into Savinase at the S135 site by yeast homologous recombination. High-titer yeast libraries were enriched by overnight cultivation at 250 rpm at 30°C in selective galactose liquid medium to remove intein-modified Savinase variants that constitutively splice at 30°C. Mutants whose inteins were not spliced at 30°C were able to grow as colonies on galactose agar plates.

Individual colonies were grown to saturation in selective glucose liquid medium (30°C, 48 h), and small aliquots (2.5 μL) were transferred to 96-well or 384-well plates containing selective galactose medium (100 μL, in duplicate) and incubated at 20°C and 37°C for 4 days.

Yeast grown in galactose medium at 20°C and 30°C were monitored at 48, 72 and 96 hours, and candidates that grew slowly at 20°C were picked from the corresponding glucose plates.

Yeast growth assay

Twelve constructs (from six inteins located at the S135 and S317 sites of Savinase: Hma_TopA, Hwa_RIR1-1, Kra_DnaB, Pho_IF2, Pho_r-Gyr, and Unc-ERS_RIR1) were first tested in E. coli, of which three (the inteins Pho_IF2, Pho_r-Gyr, and Unc-ERS_RIR1 at the S317 site) exhibited splicing activity. When expressed in yeast, these three constructs inhibited yeast growth in selective galactose medium, on agar plates, or in liquid culture, while the other constructs did not inhibit yeast growth, indicating that spliced Savinase is toxic to yeast.

Yeast growth assay was used to evaluate native inteins. Eight transformants from 314 yeast expression constructs were grown to saturation in selective glucose medium (100 μL, 96-well plate, 48 hours at 30°C). 2.5 μL aliquots were inoculated into duplicate 96-well plates containing 100 μL of selective galactose medium. One set was grown at 30°C for 72 hours and the other at 20°C for 96 hours. OD590nm was measured daily for all samples.

The same evaluation was performed on the mutagenized intein mutants. Transformants on the galactose library plates were selected by hand or colony picker and cultured in selective glucose medium (100 μL, 30°C, 48 hours). A 2.5 μL aliquot was inoculated into duplicate 96-well plates containing 100 μL of selective galactose medium, one group was grown at 30°C for 96 hours and the other group was grown at 37°C for 96 hours. Colonies that grew slowly or not at all at 20°C but grew normally at 30°C were selected as preliminary candidates. The slow growth phenotype was confirmed by repeated growth experiments in galactose medium.

Savinase activity assay based on yeast cell lysate

8 colonies from 314 NICs were cultured to saturation in non-selective glucose medium (200 μL, 96-well plate, 30°C, 48 hours). After centrifugation (3300 rpm, 5 minutes), 1 mL of selective galactose medium was added to each pellet, resuspended and cultured at 30°C for 6 hours to induce the production of recombinant protein. Cells were harvested (at 37°C, 3300 rpm, 5 minutes) and resuspended in 30 μL lysis buffer (Cellytic Y cell lysis reagent purchased from Sigma supplemented with 15 units/ml of Zymolyase, 37°C, 1 hour).

200 μL of BR buffer (pH 9.0) was added to each sample, and 40 μL of the lysate was heat-treated at 20°C or 30°C for 2 hours before the addition of succinyl-FAAF-pNA substrate and incubated at 37°C for 1 hour. After clarification by centrifugation (4500 rpm, 5 minutes), the supernatant was used for OD400nm measurement.

Savinase activity assay based on protein synthesized in vitro

The best performing candidates in the total cell lysate assay were PCR amplified and synthesized into NIC protein using a single-tube transcription and translation system (PURExpress, NEB). The synthesized NIC protein was heat-treated at 20°C and 37°C for 2 hours before adding the pNA substrate and incubating at 37°C for 30 minutes. The time course of Savinase activity was subsequently determined by OD _400nm measurement.

To investigate concentration-dependent inhibition of intein splicing by detergent, synthetic NICs, unmodified proSavinase, and inactive Savinase (H62A) were mixed with detergent to final concentrations of 25%, 5%, 1%, and 0.2%, respectively, and maintained at 20°C or 37°C for 2 hours to induce splicing. The kinetics of Savinase activity were then measured using the succinyl-FAAF-pNA substrate (37°C, 1.5 hours).

Detergent-dilution intein splicing

Similar to the detergent inhibition assay described above, Savinase from various constructs was synthesized, mixed with detergent to a final detergent concentration of 25%, and then diluted 10× with _H₂O or BR buffer (pH 9.0) to a final detergent concentration of 2.5%. Replicates were maintained at 20°C or 37°C for 2 hours before incubation with succinyl-FAAF-pNA substrate at 37°C for 90 minutes to induce splicing. OD₄₀₀ was measured at 1.0 minute intervals.

sequence:

See the sequences in the Sequence Listing filed with this application, which is incorporated herein by reference as if fully set forth.

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References cited throughout this application and the references themselves are incorporated herein for all apparent purposes, as if each reference were fully set forth. For the purpose of introduction, specific references in these references are cited at specific locations in this article. References cited at specific locations are incorporated herein in the manner taught by that reference. However, citing a reference at a specific location does not limit the incorporation of all teachings of the reference cited for any purpose into this article.

It should be understood, therefore, that the invention is not limited to the particular embodiments disclosed, but is intended to cover all modifications within the spirit and scope of the present invention as defined by the appended claims, the foregoing description and/or shown in the accompanying drawings.

Claims (17)

1. A formulation comprising a first portion and a second portion containing a peptide-modified protease and a detergent; The first part has an N-exposed peptide of the target protease and an N-include peptide of the include peptide, wherein the carboxyl terminus of the N-exposed peptide is fused to the amino terminus of the N-include peptide, and the sequence of the first part is as shown in SEQ ID NO:464. The second part comprises a C-inteptide containing an inteptide and a C-exact peptide of a target protease, wherein the carboxyl terminus of the C-inteptide is fused to the amino terminus of the C-exact peptide, and the sequence of the second part is as shown in SEQ ID NO:484; and The detergent inhibits the trans-splicing of peptide-modified proteases, and by diluting the detergent with water, the peptides can induce trans-splicing of peptide-modified proteases. The concentration of detergent in a detergent diluted with water is such that the detergent-to-water ratio is less than or equal to 1:5. 2. The formulation according to claim 1, wherein the induced concentration of the detergent in the detergent diluted with water is such that the detergent:water ratio is less than or equal to one selected from the group consisting of: 1:10, 1:20, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350 and 1:400. 3. An expression cassette comprising a polynucleotide encoding at least one of the following: i) a first portion of an inteptide-modified protease having an N-exact peptide of a target protease and an N-inteptide of an inteptide, wherein the carboxyl terminus of the N-exact peptide is fused to the amino terminus of the N-inteptide, wherein the sequence encoding the first portion is as shown in SEQ ID NO: 597; and ii) a second portion of an inteptide-modified protease having a C-inteptide of an inteptide and a C-exact peptide of a target protease, wherein the carboxyl terminus of the C-inteptide is fused to the amino terminus of the C-exact peptide, wherein the sequence encoding the second portion is as shown in SEQ ID NO: 617; and The intrinated peptide can induce trans-splicing of the protease that modifies the intrinated peptide. The ability of the intrinated peptide to induce trans-splicing is inhibited by the detergent and is restored when the detergent is diluted with water. The concentration of the detergent in the water-diluted detergent is less than or equal to 1:5 in the ratio of detergent to water. 4. A host genetically engineered to express a first or second portion of an integrin-modified protease contained in the formulation of claim 1 or 2, wherein the host is selected from the group consisting of microbial cells, mammalian cells, and insect cells. 5. The host according to claim 4, wherein the host is selected from fungal cells. 6. The host according to claim 4 or 5, wherein the host is a microorganism selected from the group consisting of Bacillus subtilus, Bacillus lentus, Bacillus licheniformis, Escherichia coli, Saccharomyces ssp., and Pichia ssp. 7. The host according to claim 6, wherein the host is Saccharomyces cerevisiae. 8. The host according to claim 6, wherein the host is Pichia pastoris. 9. A method for regulating protease activity, the method comprising exposing a peptide-modified protease to an inducing concentration of a detergent diluted with water to induce splicing of the peptide-modified protease, wherein the ratio of detergent to water is less than or equal to 1:5; Wherein, the peptide-modified protease is the protease containing peptide modification in the formulation of claim 1 or 2. 10. The method of claim 9, further comprising obtaining the peptide-modified protease. 11. The method of claim 10, wherein the obtaining step comprises genetically engineering the host by transforming the host using an expression cassette encoding an intapeptide-modified protease. 12. The method of claim 9, wherein the target protease is restored to activity via splicing of the peptide-modified protease. 13. The method of claim 11, wherein the host is selected from the group consisting of microbial cells, mammalian cells, and insect cells. 14. The method according to claim 13, wherein the microbial cells are selected from fungal cells. 15. The method according to claim 13 or 14, wherein the host is a microorganism selected from the group consisting of Bacillus subtilus, Bacillus lentus, Bacillus licheniformis, Escherichia coli, Saccharomyces ssp., and Pichia ssp. 16. The method according to claim 15, wherein the host is Saccharomyces cerevisiae. 17. The method according to claim 15, wherein the host is Pichia pastoris.