[go: up one dir, main page]

WO2004035771A1 - Acide nucleique mute d'une endonuclease cel i et methode d'obtention de la proteine cel i pleine longueur de recombinaison - Google Patents

Acide nucleique mute d'une endonuclease cel i et methode d'obtention de la proteine cel i pleine longueur de recombinaison Download PDF

Info

Publication number
WO2004035771A1
WO2004035771A1 PCT/EP2003/011210 EP0311210W WO2004035771A1 WO 2004035771 A1 WO2004035771 A1 WO 2004035771A1 EP 0311210 W EP0311210 W EP 0311210W WO 2004035771 A1 WO2004035771 A1 WO 2004035771A1
Authority
WO
WIPO (PCT)
Prior art keywords
cel
sequence
protein
dna
expression
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2003/011210
Other languages
English (en)
Inventor
Udo Baron
Ulrike Imhoff
Jürgen Koch
Marco Leuer
Jürgen Weber
Klaus Olek
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BIOPSYTEC ANALYTIK GmbH
Original Assignee
BIOPSYTEC ANALYTIK GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by BIOPSYTEC ANALYTIK GmbH filed Critical BIOPSYTEC ANALYTIK GmbH
Priority to AU2003268932A priority Critical patent/AU2003268932A1/en
Publication of WO2004035771A1 publication Critical patent/WO2004035771A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]

Definitions

  • the invention relates to a method for producing a recombinant, complete CEL I-Protein, a plant endonuclease, or parts thereof, by the expression of synthetic DNA sequences.
  • the invention also relates the DNA sequences themselves, which are produced for this purpose.
  • the invention relates to the use of the recombinantly produced CEL I-enzyme for detecting point mutations as well as larger mutated regions like e.g. deletions/insertions.
  • the CEL I-enzyme is an endonuclease found in celery (Oleykowski et al. 1998), which recognises single "uneven elements” within the DNA-double helix and cleaves there in a specific manner.
  • the enzyme therefore constitutes a very useful means for detecting mutations. It also specifically recognises single base mismatches (point mutations) and cleaves at one of the two DNA-strands proximately 3' from the mutation, thereby incising into the double strand.
  • CEL I provides a number of advantages in comparison to other known nucleases also being able to incise into DNA-strands at uneven elements of the double helix structure:
  • mismatch-recognising endonucleases Some of which are also commercially available, is based on their incomplete capability to identify all possible types of mismatches or mutations and on an unspecific DNA-degradation produced by them.
  • the SI nucleases do not cut at singles base mismatches (Loeb and Silber, 1981).
  • the Mung ⁇ e ⁇ «-Nuclease provides an efficiency being five times higher at a pH of 5, than e.g. in the neutral pH-range (Kowalski and Sandford, 1982).
  • the T4- Endonuclease VII does not only cut one strand of a double helix, but always cuts the complementary strand as well (Solaro et al., 1993). Furthermore the endonuclease isolated from T4-phages was shown to provide an unspecific activity of random DNA-degradation being significantly higher than that of the CEL I-enzyme, which is synthesised by the present method (Cotton et al., 1999). Moreover, the degree of specificity of T4-endonuclease VII exhibits a high dependence on the length of the substrate and also on the sequence surroundings of the mismatches to be detected (Babon et al., 1999; Norberg et al., 2001).
  • CEL I belongs to a distinct group of nucleases, which are found in many plant species and which are especially characterised by their specific maximum of activity at neutral pH value (Oleykowski et al., 1998), although an activity of CEL I is also to be found in a range of pH values between pH 5 and pH 9,5 (Oleykowski et al., 1998).
  • the capability to recognise and cut each form of base mismatches also irrespective of the AT- content in the vicinity of the mutation distinguishes the isolated CEL I-enzyme from other nucleases belonging to the family of plant nucleases and having their activity peak at a neutral pH value, like e.g. SP nuclease isolated from spinach (Yang et al., 2000; Oleykowski et al., 1999).
  • This objective of the invention is achieved by a special modification of the common DNA sequence of Cel I, this modification being especially designed for this aim.
  • a further aspect of the present invention is thus this newly designed sequence.
  • a further aspect of the present invention refers to a method for producing the recombinant, complete CEL I-protein comprising the following steps: At first, a scheme of the DNA- sequence to be synthesised is created. This scheme is based on the cDNA sequence of the CEL I-protein isolated from the celery plant Apium graveolens L. (see Olekowski et al., 2000).
  • the invention thus relates to a method for producing a nucleic acid sequence, which codes for the complete CEL I-protein and allows to be recombinantly expressed in host cells, whereat the method comprises the following steps: providing the sequence coding for the CEL I-protein from a suitable organism, in particular from Apium graveolens L., and adequately modifying the codon frequency of the sequence to be expressed in comparison to the native sequence, whereat this modification is performed with regard to the host organism to be used for expression.
  • This modification is accomplished by the following steps: a) partition of the planned sequence into an even number x, in particular 8, overlapping regions, b) synthesis of 2x mutated oligonucleotides, in particular oligonucleotides 1-16 to 16-16, each of which comprises the entire length of one overlapping region of both strands of the coding sequence, c) first PCR-amplification in order to produce x/2, in particular 4, overlapping fragments under employment of the oligonucleotides of step b), d) second PCR-amplification in order to produce x/4, in particular 2, overlapping regions under employment of the fragments of step c), and, e) third PCR-amplification in order to produce x/8, in particular 1, fragment, which comprises the coding region of CEL I.
  • FIG. 3 A schematic overview of a preferred embodiment is depicted in FIG. 3.
  • a method according to the invention may also be characterised in that instead of step e) the following steps are performed: e') Cloning the fragments generated in step d) into a suitable vector, and f) appropriately digesting the vectors and ligating the fragments in order to fuse them to form a complete "fragment" comprising the coding region of CEL I.
  • step e the following steps are performed: e') Cloning the fragments generated in step d) into a suitable vector, and f) appropriately digesting the vectors and ligating the fragments in order to fuse them to form a complete "fragment" comprising the coding region of CEL I.
  • a method according to the invention which is characterised by the further attachment of nucleotides coding for present/additional N-terminal or C-terminal amino acid-tags, in particular for tags being comprised of 6 histidines.
  • a base sequence in the form of a "His tag” can be added C-terminally or, in another case, N- tenninally to the original sequence, this sequence coding for 6 histidines. Due to their strong affinity to nickel ions these tags are intended to support the purification of the expressed protein by means of immobilised nickel molecules, if desired. Paula de Mattos Areas et al, e.g.
  • the fragment being provided with a His-tag sequence at its N-terminus, has a cleavage side for factor Xa 3' from the tag sequence. This allows to subsequently process the expressed enzyme by cutting off the His-tag sequence being potentially obstructive for enzyme activity.
  • the sequence designed such will be designated in the following as 6His-Xa-Cel I sequence.
  • the fragment being C-terminally linked to the His-tag will be designated as Cel I-6His in the following description of the present invention.
  • the oligonucleotides synthesised in step (c) have an average length of 70 nucleotides and overlap in each case at about 20 bases. These values however, have to be understood as mere clues and may vary in dependence of the intended use. Suitable variants are easy to find for the expert on the basis of reactive or kinetic parameters; they are particularly dependent on the temperature and the base sequence.
  • a further aspect of the invention relates to a method for producing a recombinant, complete CEL I-protein from Apium graveolens L., comprising a) performing the above described method and b) expressing the nucleic acid sequence by means of a suitable expression system.
  • a suitable expression system Especially preferred for this aim are vector-based expression systems being selected from the pPIC 9, pPIC 3, 5 and pQE-vectors.
  • vector-based expression systems being selected from the pPIC 9, pPIC 3, 5 and pQE-vectors.
  • a preferred option thereby is the expression of nucleic acid sequences in a host cell, which is selected from Hansenula polymorpha, Pichia pastoris, Saccharomyces cerevisiae, HeLa- cells, CHO-cells, Toxoplasma gondii and Leishmania.
  • a host cell which is selected from Hansenula polymorpha, Pichia pastoris, Saccharomyces cerevisiae, HeLa- cells, CHO-cells, Toxoplasma gondii and Leishmania.
  • a method, in which the employed Pichia pastoris strain is the stain GS115.
  • the invention also relates to the complete DNA sequence of the CEL I-protein or expressible parts thereof derived from Apium graveolens, whereat said sequence is adapted for expression and is provided by a method according to the present invention.
  • the wording "expressible parts” thereof refers to parts of the nucleic acid sequence coding for a polypeptide chain having an enzymatic function, in particular the function of the native CEL I-enzyme. Also comprised by the scope of the invention however, are nucleic acids coding for epitopes.
  • a preferred form is represented by a DNA sequence according to the invention, which codes for the Apium graveolens CEL I-protein, this sequence being characterised in that furthermore nucleotides are added, which encode additional N-terminal or C-terminal amino acid-tags, in particular tags being comprised of 6 histidines.
  • the sequence at its both ends is equipped with restriction endonuclease cleavage sites, which are absent in the remaining sequence and in a vector to be employed.
  • a most preferred Apium graveolens CEL I-protein encoding sequence according to the invention or parts thereof is presented in SEQ ID No.7. "Parts" in this context especially mean the fragments serving as probes, but also the overlapping oligonucleotides used for generating and cloning (see FIG. 2).
  • Both CEL I coding sequence variants were equipped at their ends with sequences of restriction sites, which are helpful for subsequent cloning steps. Examples for these sequences are the EcoRI restriction site positioned at both ends or the Xhol restriction site at the C- terminus (FIG. 2). Moreover, the translational start ATG was integrated in a Kozak sequence (consensus sequence for translational initiation) (ACC ATG G) (Kozak 1987; Kozak 1990) in both sequences. In the further procedure, 16 deoxyoligonucleotides were synthesised, which correspond to the planned sequence and completely cover the whole length of the respective cDNA.
  • the deoxyoligonucleotides were synthesised such, that their sequences were alternatively corresponding to the 5 '-3'- or to the complementary 3'-5'-DNA-strand.
  • the length of the deoxyoligonucleotides was between 40 to 93 bases with overlaps of an average of 20 bases between neighbouring sequences (FIG. 2).
  • the artificial CEL I-gene was synthesised in the form of two independent partial fragments, the N-terminal and the C- terminal fragment, and fused afterwards via a HindM restriction site (FIG. 2).
  • the generation of a partial fragment was accomplished according to the following principle: In a first step, four DNA sequences having the double length of two neighbouring deoxyoligonucleotides (minus the overlapping sequences) were generated for each fragment by means of asymmetric PCR. The amplification was accomplished such, that the neighbouring, accumulated DNA-strands in each case represent the opposite strand.
  • the artificial CEL I-gene generated in such a way can be transferred into suitable expression vectors during subsequent steps of the procedure.
  • Favourable examples for these vectors are - among others - expression vectors suitable for the Pichia pastoris expression system like the pPIC 9 or the pPIC 3, 5 vector (Invitrogen).
  • other expression vectors and host organisms other than yeast which are familiar to the expert.
  • the subject of the invention is thus not restricted to a special host system.
  • a further aspect of the present invention thus relates to a host organism, which is capable to integrate and express a DNA sequence according to the invention.
  • This host preferably is selected from Hansenula polymorpha, Pichia pastoris, Saccharomyces cerevisiae, HeLa- cells, CHO-cells, Toxoplasma gondii and Leishm ⁇ ni ⁇ .
  • Pichia pastoris stem GS115 is used.
  • plant cells or insect cells may also be employed.
  • a preferred host organism in this invention which is employed for expression, is the yeast Pichia pastoris (Invitrogen), whereof the preferred yeast strain is GS115 (Invitrogen).
  • Yeast in general is preferred as the expression system, since it has - as a eukaryotic organism - many advantages compared to bacterial systems for expression, like e.g. the post-translational processing of proteins.
  • Another important advantage of using a eukaryotic expression system is based on the cellular compartmentation being present in eukaryotic organisms.
  • nucleases Expressing nucleases by means of recombinant expression systems in prokaryotic host organisms like bacteria is toxic for the cells due to the nucleases' DNA-degrading properties and has consequently and several times been described as being extremely difficult (Golz et a., 1995; Kosak and Kemper, 1990).
  • Pichia pastoris moreover is able to metabolise methanol as a hydrocarbon source.
  • the first step of methanol catabolism is catalysed by alcohol oxidase.
  • Pichia harbours two genes, which code for this enzyme, the AOX1- and the AOX2-gene, whereat the AOXl-gene provides the by far greater portion of active alcohol oxidase in the cells.
  • the expression of the AOXl-gene is regulated and induced by methanol.
  • the AOXl-gene was isolated and the AOXl-promotor was used for the expression of an arbitrary gene (Ellis et al., 1985; Koutz et al., 1989; Tschopp et al., 1987a).
  • the form of heterologous expression of the CEL I-enzyme in Pichia pastoris being preferred in this invention is the secretory form of protein expression.
  • Secretory protein expression in Pichia pastoris has the advantage, that - because of the very low level of native protein secretion of this yeast - the major component of the total protein in the medium is constituted by the desired protein. This facilitates further steps of purification of the heterologous protein or even makes them potentially unnecessary.
  • the secretory mechanism preferably used in this expression method is based on the secretion signal ⁇ -factor of Saccharomyces cerevisiae (Barr et al., 1992), which is already integrated in the prefabricated expression vector pPIC 9 (Invitrogen).
  • Pichia pastoris A further reason for the preference of the Pichia systems e.g. to prokaryotic expression systems is the capability of Pichia pastoris to perform post-translational modification like e.g. the N-glycosidic affiliation of sugars, but without causing hyperglycosylation like it is e.g. the case with S. cerevisiae (Grinna and Tschopp, 1989; Tschopp et al., 1987b). Post-translational modifications can be crucial for the proper function of an enzyme.
  • the construct which is preferred in this invention for the expression of the active CEL I- enzyme consists of the Cel I-6His-sequence-molecule, which is ligated in the appropriate orientation into the EcoRI restriction site of the expression vector pPIC 9, and which in this form has its open reading frame in fusion with the signal peptide.
  • the C ⁇ L I-gene preferably the Cel I-6His-construct
  • the vector provides both an ampicillin resistance and an E.coli origin of replication (FIG. 4).
  • the integration in the case being preferred herein, in accomplished by a homologous recombination, i.e. by a crossing over between the His4-locus on the chromosome and the His4-locus on the vector.
  • the His4-gene of Pichia pastoris is used for the selection of stable transformants.
  • the His4-gene which is part of the histidine metabolism pathway, is present in the yeast genome in a mutated form, whereas it is present in the vector in the wildtype form.
  • yeast cells without the integrated vector are not capable to grow on histidine- free medium, whereas yeast cells successfully transformed with pPIC9 are capable to form colonies on a medium containing no histidine.
  • the vector lacks a yeast origin of replication, only yeast cell colonies can arise, in the founder cell of which a recombination has taken place between the plasmid and the yeast genome whereby the vector including the target gene has been integrated into the yeast genome.
  • the preferred technique of transformation in this method is the yeast transformation by means of electroporation. For this technique one adds 20-30 ⁇ g of linearised vector-DNA, purified by phenol extraction after linearisation, to 80 ⁇ l freshly competent cells of the yeast strain GS115 in a sterile cuvette.
  • the CEL I-6His-pPIC9 construct in this method is linearized via the unique Sail restriction site (FIG. 4).
  • the electroporation was performed by means of a Gene Pulser II Systems (Biorad) employing 50 ⁇ F/200 ⁇ /l,8V and a pulse time of about 10 msec.
  • Successfully transformed cells can be identified after an incubation period of 5 days on a histidine-free medium as properly grown colonies. Further evaluation in respect of a stable transformation was accomplished by a PCR-based detection of the target gene within the yeast genome.
  • clones of 20 yeast clones tested were identified as unambiguously positive (FIG. 5).
  • PCR-positive clones were analysed by hybridising a respective Southern Blot with a CEL I-specific probe.
  • controls used were a plasmid-DNA of the CEL I-6His-pPIC9-construct (positive control) and a genomic DNA being transformed with the parental vector pPIC9 without the CEL I-insert into yeast (negative control).
  • a digoxigenin-labelled probe with a length of 262 bases was synthesised.
  • the probe synthesis was accomplished by means of two oligonucleotides specifically annealing in the N-terminal region of the coding CEL I- sequence, the oligonucleotides "Sonde f" ("probe f") 5'-
  • ATGACCAGACTGTACTCCGTGTTC-3' (SEQ ID No. 3) and "Sonde r" (“probe r") 5'- GTCAGGGGTATCAATGAAATGTAA-3' (SEQ ID No.4; FIG. 2).
  • a further aspect of the invention refers to a recombinant, complete CEL I-protein produced by a method according to the invention.
  • the desired enzyme was able to be easily purified from the supernatant of the expression culture by means of techniques known in the prior art. After having concentrated the proteins in the supernatant by a factor of about 200 by means of ultrafiltration tubes (Vivascience), the active CEL I-enzyme allowed to be used directly as a protein, which recognises and cleaves mismatch sequences.
  • constructs were created for this purpose, which allowed to synthesise all of the eight mismatch combinations by means of the respective combinations of heterohybrids.
  • the generation of these constructs was accomplished by the cloning of four oligonucleotides into the EcoRI/Hindlll cleaved pUC19 vector. These oligonucleotides only differed at one single base position (FIG. 7).
  • the four cloned fragments were able to be used as defined templates for the amplification of fragments using fluorescence-labelled oligonucleotides directly taking part in heterohybrid formation. According to the combination of amplification targets in hetero-hybrid synthesis, all of the eight mismatch combinations possible allowed to be generated.
  • the amplification of 237 bp fragments was accomplished by means of the fluorescence- labelled PUC19 F-primer 5'-FAM-GGATGTGCTGCAAGGCGAT-3' (SEQ ID No.5) and the fluorescence-labelled PUC19 R-primer 5'-JOE-GTGAGTTAGCTCACTCATTAG-3' (SEQ ID No.6).
  • the activity assay was performed by incubating the heterohybrids with a 1 :50 dilution of the CEL I-extract from the yeast expression supernatant at 47°C for 10 min.
  • the enzyme produced in this method by means of the artificially synthesised gene exactly displays its desired property, i.e. the precise recognition of all possible mismatch combinations as well as the subsequent incision into one strand at the phosphodiester bond immediately 3' of the detected base mismatch (Leykowski et al., 1998).
  • a further aspect thus relates to the use of the recombinantly produced CEL I-enzyme according to the invention for detecting both point mutations as well as larger mutated regions like e.g. deletions/insertions.
  • FIG. 1 A depiction of the nucleic acid sequence (SEQ ID No. 7) required for encoding the mature CEL I-enzyme after redraft for expressing the enzyme in yeast.
  • the amino acid sequence (SEQ ID No. 8) is also presented. Furthermore presented are base deviations from the published original sequence, which are shown in grey characters.
  • FIG. 2 A depiction of the complete nucleotide sequence of the synthetic CEL I-gene (SEQ ID No. 9). The sequence is given as a double strand, whereat the deoxyoligonucleotides necessary for synthesis are each printed in boldface on the respective strand. Moreover indicated are the sequence modifications like restriction sites, the Kozak sequence and the His-tag encoding sequences. The His-tag encoding sequence sections positioned at the N- terminus or the C-terminus are underlined; the underlined sequences were added either N- terminally or C-terminally, but not at both termini.
  • FIG. 3 Schematic depiction concerning the synthesis of the artificial CEL I-gene by means of asymmetric PCR employing 16 overlapping deoxyoligonucleotides.
  • FIG. 4 Schematic depiction of the vector pPIC9 (Invitrogen), which is preferably used in this invention.
  • the figure shows the integration of the artificial CEL I-gene (about 100 bp) into the EcoRI-restriction site of the vector; also shown is the oligonucleotide-primer AOX3' required for the PCR-test.
  • FIG. 5 PCR-result for the verification of the integration of the C ⁇ L I-gene into the genome of several yeast clones.
  • Genomic DNA as a template was isolated from 20 yeast clones to be tested.
  • Genomic DNA of two non-transformed yeast clones served as a template for the negative control (-).
  • Purified vector-DNA of two original constructs served as a template for the positive control.
  • the blank sample comprised water in order to exclude contaminations.
  • 16 clones of 20 clones to be tested are unambiguously positive. Corresponding to the positive controls, they show a band of about 1000 bp. Negative controls and blank sample are free of this signal. As a molecular weight marker (M) the "1 kb Plus DNA Ladder" (Gibco) is shown.
  • FIG. 6 Result of the Southern hybridisation for the further verification of 12 yeast clones tested before by the PCR method. Both as a positive control and as a size control, the 262 bp C ⁇ L I-specific probe was hybridised to plasmid-DNA (+) (Construct shown in FIG. 4).
  • the probe was hybridized to genomic yeast DNA of a clone containing the parental vector pPIC 9 without the C ⁇ L I-insert (-).
  • FIG. 7 Construct for generating defined heterohybrids for performing a specific activity assay of the C ⁇ L I-enzyme.
  • the two depicted synthetic oligonucleotides are constructed such, that they can be directly ligated into the EcoRL/Hindlll digested pUC19-vector after annealing (what is possible due to their complementary nature).
  • Each of the two deoxyoligonucleotides is present in fourfold version.
  • the letters Y and Z each symbolise all of the four possible bases. In consequence, all of the eight possible base mismatches (AA/TT/CC/GG/AC/AG/TC/TG) can be synthesised depending on the combination of the oligonucleotides.
  • Grinna L S and Tschopp J F Size distribution and general structural features of N-linked oligosaccarides from the methylotrophic yeast Pichia pastoris. Yeast 5: 107-115 (1989)
  • Tschopp J F, House P F, Cregg J M, Stillmann C and Gingeras T R Expression of the lacZ gene from two methanol regulated promoters in Pichia pastoris.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne une méthode d'obtention d'une protéine CEL-I complète de recombinaison, d'une endonucléase végétale et de parties de ces dernières par expression de séquences d'ADN synthétique. L'invention concerne également les séquences d'ADN elles-mêmes produites à cette fin. De plus, l'invention porte sur une enzyme CEL-I obtenue par recombinaison qui permet de détecter des mutations ponctuelles ainsi que des régions mutées plus importantes telles que des déletions/insertions.
PCT/EP2003/011210 2002-10-16 2003-10-09 Acide nucleique mute d'une endonuclease cel i et methode d'obtention de la proteine cel i pleine longueur de recombinaison Ceased WO2004035771A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003268932A AU2003268932A1 (en) 2002-10-16 2003-10-09 Mutated nucleic acid of a cel i-endonuclease and method for producing the recombinant, full-length cel i-protein

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10248258.6 2002-10-16
DE2002148258 DE10248258A1 (de) 2002-10-16 2002-10-16 Mutierte Nukleinsäure einer Cel I-Endonuklease und Verfahren zur Herstellung des rekombinanten vollständigen Cel I-Proteins

Publications (1)

Publication Number Publication Date
WO2004035771A1 true WO2004035771A1 (fr) 2004-04-29

Family

ID=32086946

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2003/011210 Ceased WO2004035771A1 (fr) 2002-10-16 2003-10-09 Acide nucleique mute d'une endonuclease cel i et methode d'obtention de la proteine cel i pleine longueur de recombinaison

Country Status (3)

Country Link
AU (1) AU2003268932A1 (fr)
DE (1) DE10248258A1 (fr)
WO (1) WO2004035771A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1565557A4 (fr) * 2002-02-01 2005-11-30 Large Scale Biology Corp Endonucleases non appariees et leurs procedes d'utilisation
WO2006010646A1 (fr) * 2004-07-30 2006-02-02 Genoplante-Valor Procede de production d'endonucleases a sensibilite elevee, nouvelles preparations d'endonucleases et leurs utilisations
US7217514B2 (en) 2001-02-02 2007-05-15 Large Scale Biology Corporation Method of increasing complementarity in a heteroduplex
US7838219B2 (en) 2001-02-02 2010-11-23 Novici Biotech Llc Method of increasing complementarity in a heteroduplex
US20130225451A1 (en) * 2012-02-01 2013-08-29 Synthetic Genomics, Inc. Materials and methods for the synthesis of error-minimized nucleic acid molecules

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997046701A1 (fr) * 1996-06-05 1997-12-11 Fox Chase Cancer Center Endonucleases sensibles au mesappariement et leurs utilisations pour la detection de mutations dans des brins de polynucleotides cibles
WO2001062974A1 (fr) * 2000-02-22 2001-08-30 Fox Chase Cancer Center Molecule d'acide nucleique codant pour une endonuclease mesapariee et ses procedes d'utilisation
WO2001066693A1 (fr) * 2000-03-10 2001-09-13 Novozymes A/S Compositions et procedes permettant de produire des polypeptides heterologues haut rendement dans une cellule de pichia

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4808537A (en) * 1984-10-30 1989-02-28 Phillips Petroleum Company Methanol inducible genes obtained from pichia and methods of use

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997046701A1 (fr) * 1996-06-05 1997-12-11 Fox Chase Cancer Center Endonucleases sensibles au mesappariement et leurs utilisations pour la detection de mutations dans des brins de polynucleotides cibles
WO2001062974A1 (fr) * 2000-02-22 2001-08-30 Fox Chase Cancer Center Molecule d'acide nucleique codant pour une endonuclease mesapariee et ses procedes d'utilisation
WO2001066693A1 (fr) * 2000-03-10 2001-09-13 Novozymes A/S Compositions et procedes permettant de produire des polypeptides heterologues haut rendement dans une cellule de pichia

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
OLEYKOWSKI C A ET AL: "MUTATION DETECTION USING A NOVEL PLANT ENDONUCLEASE", NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 26, no. 20, 1998, pages 4597 - 4602, XP002943289, ISSN: 0305-1048 *
OUTCHKOUROV NIKOLAY S ET AL: "Optimization of the expression of equistatin in Pichia pastoris", PROTEIN EXPRESSION AND PURIFICATION, vol. 24, no. 1, February 2002 (2002-02-01), pages 18 - 24, XP002266045, ISSN: 1046-5928 *
SINCLAIR GRAHAM ET AL: "Synonymous codon usage bias and the expression of human glucocerebrosidase in the methylotrophic yeast, Pichia pastoris.", PROTEIN EXPRESSION AND PURIFICATION, vol. 26, no. 1, October 2002 (2002-10-01), pages 96 - 105, XP002266046, ISSN: 1046-5928 *
WOO JUNG HEE ET AL: "Gene optimization is necessary to express a bivalent anti-human anti-T cell immunotoxin in Pichia pastoris", PROTEIN EXPRESSION AND PURIFICATION, vol. 25, no. 2, July 2002 (2002-07-01), pages 270 - 282, XP002266044, ISSN: 1046-5928 *
YANG BING ET AL: "Purification, cloning, and characterization of the CEL I nuclease", BIOCHEMISTRY, AMERICAN CHEMICAL SOCIETY. EASTON, PA, US, vol. 39, no. 13, 4 April 2000 (2000-04-04), pages 3533 - 3541, XP002222862, ISSN: 0006-2960 *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7833759B2 (en) 2001-02-02 2010-11-16 Novici Biotech Llc Method of increasing complementarity in a heteroduplex
US7838219B2 (en) 2001-02-02 2010-11-23 Novici Biotech Llc Method of increasing complementarity in a heteroduplex
US7235386B2 (en) 2001-02-02 2007-06-26 Large Scale Biology Corporation Method of increasing complementarity in a heteroduplex
US7217514B2 (en) 2001-02-02 2007-05-15 Large Scale Biology Corporation Method of increasing complementarity in a heteroduplex
US7078211B2 (en) 2002-02-01 2006-07-18 Large Scale Biology Corporation Nucleic acid molecules encoding endonucleases and methods of use thereof
US7056740B2 (en) 2002-02-01 2006-06-06 Large Scale Biology Corporation Mismatch endonucleases and methods of use
US7273739B2 (en) 2002-02-01 2007-09-25 Padgett Hal S Nucleic acid molecules encoding endonucleases and methods of use thereof
EP1565557A4 (fr) * 2002-02-01 2005-11-30 Large Scale Biology Corp Endonucleases non appariees et leurs procedes d'utilisation
WO2006010646A1 (fr) * 2004-07-30 2006-02-02 Genoplante-Valor Procede de production d'endonucleases a sensibilite elevee, nouvelles preparations d'endonucleases et leurs utilisations
JP2008507965A (ja) * 2004-07-30 2008-03-21 ジュノプラント−ヴァロール 高感受性エンドヌクレアーゼを生産する方法、新規なエンドヌクレアーゼ調製物、及びそれらの使用
AU2005266465B2 (en) * 2004-07-30 2011-01-20 Genoplante-Valor Method for producing highly sensitive endonucleases, novel preparations of endonucleases and uses thereof
JP4836952B2 (ja) * 2004-07-30 2011-12-14 ジュノプラント−ヴァロール 高感受性エンドヌクレアーゼを生産する方法、新規なエンドヌクレアーゼ調製物、及びそれらの使用
US20130225451A1 (en) * 2012-02-01 2013-08-29 Synthetic Genomics, Inc. Materials and methods for the synthesis of error-minimized nucleic acid molecules
US9771576B2 (en) 2012-02-01 2017-09-26 Synthetic Genomics, Inc. Materials and methods for the synthesis of error-minimized nucleic acid molecules
US10704041B2 (en) 2012-02-01 2020-07-07 Codex Dna, Inc. Materials and methods for the synthesis of error-minimized nucleic acid molecules
US11884916B2 (en) 2012-02-01 2024-01-30 Telesis Bio Inc. Materials and methods for the synthesis of error-minimized nucleic acid molecules

Also Published As

Publication number Publication date
DE10248258A1 (de) 2004-05-06
AU2003268932A1 (en) 2004-05-04

Similar Documents

Publication Publication Date Title
AU2017272206B2 (en) Materials and methods for the synthesis of error-minimized nucleic acid molecules
EP2106447B1 (fr) Procédé pour l'induction indépendante du méthanol à partir de promoteurs inductibles par le méthanol dans pichia
CA2210242C (fr) Production de carboxypeptidase b recombinee a activite enzymatique
JP6910358B2 (ja) 酵母細胞
WO2011030347A1 (fr) Nouvelles protéines de fusion prolipase-trypsinogène bovin
JP3236862B2 (ja) アスペルギルス・ニガーのカルボキシペプチダーゼをコードする遺伝子
CN103710317A (zh) 一种漆酶突变体及其编码基因与应用
WO2004035771A1 (fr) Acide nucleique mute d'une endonuclease cel i et methode d'obtention de la proteine cel i pleine longueur de recombinaison
EP1902138B1 (fr) Promoteurs thermoinductibles de la tetrahymena et leur utilisation
US6699691B2 (en) Alcohol oxidase 1 regulatory nucleotide sequences for heterologous gene expression in yeast
US7718398B2 (en) Promoters having a modified transcription efficiency and derived from methyltrophic yeast
WO2018196881A1 (fr) Glucose oxydase cngoda, gène et application associés
JPWO2008032659A1 (ja) 効率向上型分泌シグナルペプチド及びそれらを利用したタンパク質生産方法
CN120077141A (zh) 修饰的启动子序列
CA2381347A1 (fr) Sequences regulatrices et cassettes d'expression pour les levures
JP4413557B2 (ja) 糸状菌を用いたタンパク質の効率的製造法
JP7756787B2 (ja) タンパク質合成および分泌のための方法および組成物
JP3549551B2 (ja) Ｓ．セレビシエのリボフラビンシンテターゼ活性をコードするｄｎａ化合物および組換えｄｎａ発現ベクター
JP4671394B2 (ja) キャンディダ・ユティリス由来のプロモーターｄｎａ
US20250019683A1 (en) Tritirachium album proteinase k mutant and its zymogen, expression plasmid, recombinant pichia pastoris strain and method of producing the mature form of proteinase k mutant
AU7062700A (en) D-gluconolactone oxidase gene and method for producing recombinant d-gluconolactone oxidase
CN119899865A (zh) OsWRKY76蛋白在提高磷的利用效率和水稻产量中的应用
CN113913414A (zh) 高稳定性和高催化效率的双碱基酶Kex2突变体
JP2011167160A (ja) 新規ターミネーターおよびその利用
El-Adawi et al. Overexpression of protein disulfide isomerase in Aspergillus

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP