JP7768775B2 - Peptide tags and tagged proteins containing the same - Google Patents

Description

The present invention relates to peptide tags, tagged proteins containing the same, and DNA encoding the same.
The present invention also relates to a transformant containing said DNA and a method for producing a tagged protein.

Thanks to advances in recombinant DNA technology, the production of useful proteins through heterologous expression is now commonplace. In the production of useful proteins through heterologous expression, methods for improving protein expression and accumulation are being considered, including the selection of promoters and terminators, translation enhancers, codon modification of introduced genes, and intracellular transport and localization of proteins. For example, Patent Document 1 discloses a technique for expressing bacterial toxin proteins in plants, and discloses that bacterial toxin proteins are expressed by linking them with peptide linkers in which prolines are spaced at regular intervals (Patent Document 1).

In addition, several other technologies have been developed to improve expression by linking peptide tags to target proteins (Patent Documents 2-6, Non-Patent Documents 1-3).

Patent No. 5360727 specification Patent No. 5273438 specification International Publication No. WO2016/204198 Brochure International Publication No. WO2017/115853 Brochure International Publication No. WO2020/045530 Brochure U.S. Patent Publication No. 20090137004

Smith, D. B. and Johnson, K. S.,: Gene, 67, 31, 1988. Marblestone, J. G. et al.: Protein Sci., 15, 182, 2006 di Guan, C. et al.: Gene, 67, 21, 1988

Patent Document 1 discloses that linking toxin proteins using a peptide linker with regularly spaced prolines enables high accumulation of toxin fusion proteins in plants. Furthermore, Patent Documents 4 and 5 examine the amino acids between prolines in peptide tags and provide peptide tags suitable for high protein expression and soluble expression. However, these peptide linkers and peptide tags are based on the presence of regularly spaced prolines, and there is room for further study of the sequences to improve their performance as high-protein expression tags. Therefore, an objective of the present invention is to provide a new peptide tag that can increase the expression level of a target protein by binding to the target protein when the protein is expressed in a host cell or a cell-free expression system.

The inventors investigated sequences in an effort to improve the performance of peptide tags. They then used a peptide tag with the amino acid sequence shown in formula (1) below and examined the expression level of proteins to which it was attached. They found that the expression level of the target protein was significantly improved. The present invention was made based on these findings.

That is, the present invention is as follows.
[1] A peptide having an amino acid sequence represented by the following formula (1), (2), or (3), and having a length of 3 to 6 amino acids:
X _m Z _n U _q ...(1)
X _m Z _n U _q Z _n ...(2)
XZUZUZ...(3)
wherein X is an amino acid residue independently selected from isoleucine (I), phenylalanine (F), methionine (M), alanine (A), valine (V), arginine (R), glutamine (Q), and glutamic acid (E);
Z is an amino acid residue independently selected from lysine (K) and asparagine (N);
U is an amino acid residue independently selected from glycine (G), isoleucine (I), glutamine (Q), valine (V), histidine (H), leucine (L), alanine (A), aspartic acid (D), glutamic acid (E), arginine (R), and threonine (T);
m and n are integers of 1 or 2; q is an integer of 0, 1, 2, or 3;
[2] The peptide according to [1], which consists of 4 to 5 amino acid residues.
[3] The peptide according to [1] or [2], wherein m is 1 and q is 1 or 2.
[4] An amino acid sequence of any one of SEQ ID NOs: 1 to 20 and 71 to 81, or INK
The peptide according to any one of [1] to [3], having any one of the amino acid sequences of INE, RNK, and RND.
[5] A tagged protein comprising the peptide according to any one of [1] to [4] and a useful protein.
[6] The tagged protein according to [5], wherein the useful protein is an enzyme, cytokine, antibody, or fluorescent protein.
[7] A DNA encoding the tagged protein according to [5] or [6].
[8] A recombinant vector comprising the DNA according to [7].
[9] A transformant transformed with the DNA according to [7] or the recombinant vector according to [8].
[10] A method for producing a tagged protein, comprising culturing the transformant according to [9] to express and accumulate the tagged protein, and recovering the tagged protein.
[11] A method for producing a tagged protein, comprising introducing the DNA according to [7] or RNA transcribed therefrom into a cell-free expression system to express and accumulate the tagged protein, and recovering the tagged protein.

The use of the peptide tag of the present invention can improve the expression level of a target protein. Therefore, it is useful for producing proteins using host cells such as yeast, Escherichia coli, and Brevibacillus, or cell-free expression systems.

Schematic diagram of the construction of an E. coli expression vector for tagged proteins (with N-terminus tagging). Schematic diagram of the construction of an E. coli expression vector for tagged proteins (with C-terminus tagging). Schematic diagram of the E. coli-Yarrowia lipolytica shuttle vector. Schematic diagram of construction of tagged protein expression vector (N-terminal tag added) for Yarrowia lipolytica. 1 is a graph showing the expression level of tagged green fluorescent protein (GFP2) in a cell-free expression system, where the expression level of untagged GFP2 (Comparative Example A) is set to 1. Graph showing the expression level of N-terminal tagged GFP2 in Escherichia coli (BL21) upon IPTG induction (Examples 1 to 20). The expression level is shown as a relative value when the expression level of untagged GFP2 (Comparative Example A) is set to 1. Graphs showing the expression level of N-terminal tagged GFP2 in Escherichia coli (BL21) upon IPTG induction (Examples 21 to 24, 29 to 35). The values shown are relative to the expression level of untagged GFP2 (Comparative Example A), which is set to 1. 1 is a graph showing the fluorescence intensity of N-terminally tagged GFP2 in Escherichia coli (BL21) upon IPTG induction (Examples 1 to 20). The fluorescence intensity is shown as a relative value when the fluorescence intensity of untagged GFP2 (Comparative Example A) is set to 1. Graphs showing the fluorescence intensity of N-terminal tagged GFP2 in Escherichia coli (BL21) upon IPTG induction (Examples 21 to 24, 29 to 35). The fluorescence intensity is shown relative to the fluorescence intensity of untagged GFP2 (Comparative Example A), which is set to 1. Graphs showing the expression levels of N-terminal tagged VHH antibodies in Escherichia coli (BL21) upon IPTG induction (Examples 10, 12, and 25). The expression levels are relative to the expression level of untagged GFP2 (Comparative Example A), which is set to 1. Graphs showing the expression levels of VHH antibodies tagged at the C-terminus in Escherichia coli (BL21) upon IPTG induction (Examples 23, 29, and 31). The values shown are relative to the expression level of untagged GFP2 (Comparative Example A), which is set to 1. Graph showing the expression level of N-terminal tagged GFP2 in Yarrowia lipolytica (Examples 10, 12, 15, 18, 20, 25 to 28). The expression level of untagged GFP2 (Comparative Example A) is set to 1, and the relative values are shown.

The peptide of the present invention (also referred to as a peptide tag) has an amino acid sequence of the following formula (1), (2), or (3):
X _m Z _n U _q ...(1)
X _m Z _n U _q Z _n ...(2)
XZUZUZ...(3)

Each X is an amino acid residue independently selected from I, F, M, A, V, R, Q and E, preferably R, M, Q, E or I, more preferably R.
m is 1 or 2, preferably 1.
_Xm means that one or two consecutive Xs are present, and when m is 2, there may be two of the same amino acid residues selected from I, F, M, A, V, R, Q, and E, or there may be two different amino acid residues.

Z is K or N, and m is 1 or 2, preferably 1.
_Zn means one or two consecutive Z's, and is any of the following:
K
N
K.K.
N.N.
KN
NK
In formula (2), there are two Z _n , and _they , including _{the number} of n, may be _different . Examples include _XmNUqK , _{XmNUqN , XmNUqKN , XmNUqKK , XmNUqNN , XmKUqK} , _{XmKUqN , XmKUqKN , XmKUqKK} , and _XmKUqNN .
In formula (3), there are three Z's, which may be different from each other. Examples include XKUKUK, XKUKUN, XKUNUN, XNUNUN, XNUNUK, and XNUKUK.

Each U is an amino acid residue independently selected from G, I, Q, V, H, L, A, D, E, R, and T. For example, U is G.
U _q means q consecutive U's, and in this case, the q U's may be the same amino acid residue selected from G, I, Q, V, H, L, A, D, E, R, and T, or may be different amino acid residues.
q is 0, 1, 2 or 3, preferably 1 or 2.
In formula (3), there are two U's, which may be different.

The peptides of the present invention are 3 to 6 amino acids in length, preferably 3 to 5 amino acids in length, even more preferably 4 to 5 amino acids in length, and particularly preferably 4 amino acids in length.

Specific examples of peptides of the present invention include, but are not limited to, peptides consisting of any of the amino acid sequences of SEQ ID NOS: 1 to 20 and 71 to 81 shown in Table 1 below, or any of the amino acid sequences of INK, INE, RNK, and RND.

The tagged protein of the present invention comprises a target protein to which the peptide tag of the present invention is bound (also referred to as a fusion protein of a tag and a target protein). The peptide tag may be bound to the N-terminus of the target protein, the C-terminus of the target protein, or both the N-terminus and the C-terminus of the target protein. The peptide tag may be bound directly to the N-terminus and/or the C-terminus of the target protein, or may be bound via a sequence of one to several amino acids (e.g., one to five amino acids). The sequence of one to several amino acids may be any sequence as long as it does not adversely affect the function or expression level of the tagged protein. However, by including a protease recognition sequence, the peptide tag can be cleaved from the useful protein after expression and purification. An example of a protease recognition sequence is a factor Xa recognition sequence. The tagged protein of the present invention may also contain other tag sequences necessary for detection, purification, etc., such as a His tag, an HN tag, or a FLAG tag.

Useful proteins contained in the tagged proteins of the present invention are not particularly limited, but include growth factors, hormones, cytokines, blood proteins, enzymes, antigens, antibodies, transcription factors, receptors, fluorescent proteins, or partial peptides thereof.

Enzymes include, for example, lipases, proteases, steroidogenic enzymes, kinases, phosphatases, xylanases, esterases, methylases, demethylases, oxidases, reductases, cellulases, aromatases, collagenases, transglutaminases, glycosidases, and chitinases.

Examples of growth factors include epidermal growth factor (EGF), insulin-like growth factor (IGF), transforming growth factor (TGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), platelet-derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin (TPO), fibroblast growth factor (FGF), and hepatocyte growth factor (HGF).

Hormones include, for example, insulin, glucagon, somatostatin, growth hormone, parathyroid hormone, prolactin, leptin, and calcitonin.

Examples of cytokines include interleukins, interferons (IFNα, IFNβ, IFNγ), and tumor necrosis factor (TNF).

Examples of blood proteins include thrombin, serum albumin, factor VII, factor VIII, factor IX, factor X, and tissue plasminogen activator.

Examples of antibodies include complete antibodies, Fab, F(ab'), F(ab') ₂ , Fc, Fc fusion proteins, heavy chains (H chains), light chains (L chains), single-chain Fvs (scFv), sc(Fv) ₂ , disulfide-linked Fvs (sdFv), diabodies, and VHH antibodies.

There are no particular restrictions on the antigenic proteins used as vaccines, as long as they are capable of eliciting an immune response, and they may be selected appropriately depending on the target of the intended immune response. Examples include proteins derived from pathogenic bacteria and pathogenic viruses.

The tagged protein of the present invention may have a secretory signal peptide functional in host cells added thereto for secretory production. Examples of secretory signal peptides include an invertase secretory signal, a P3 secretory signal, and an α-factor secretory signal when yeast is used as the host; a PelB secretory signal when Escherichia coli is used as the host; and a P22 secretory signal when Brevibacillus is used as the host. Furthermore, when a plant is used as the host, examples of secretory signal peptides include those derived from plants belonging to the Solanaceae, Rosaceae, Brassicaceae, and Asteraceae families, more preferably from plants belonging to the Nicotiana , Arabidopsis , Fragaria , and Lactuca genera, such as Nicotiana tabacum, Arabidopsis thaliana , Fragaria × ananassa , and lettuce ( Lactuca sativa ).

Furthermore, the tagged proteins of the present invention may be tagged with a transport signal peptide such as an endoplasmic reticulum retention signal peptide or a vacuolar targeting signal peptide in order to express them in a specific cellular compartment.

The tagged proteins of the present invention can be chemically synthesized or produced by genetic engineering. Methods for producing them by genetic engineering are described below.

The DNA of the present invention is characterized by comprising DNA encoding the tagged protein of the present invention. That is, the DNA of the present invention comprises DNA encoding a useful protein and DNA encoding a peptide tag. The DNA encoding the useful protein and the DNA encoding the peptide tag are ligated in the same reading frame.

DNA encoding a useful protein can be obtained, for example, by general genetic engineering techniques based on a known base sequence.
Furthermore, it is also preferred that the DNA encoding the tagged protein of the present invention has codons representing the amino acids that make up the tagged protein appropriately modified to increase the translation yield of the hybrid protein, depending on the host cell in which the protein is produced. Other methods include selecting codons that are frequently used in the host cell, codons with a high GC content, or codons that are frequently used in housekeeping genes of the host cell.

The DNA of the present invention may contain an enhancer sequence that functions in host cells to improve expression in the host cells. Examples of enhancers include a Kozak sequence and the 5'-untranslated region of a plant-derived alcohol dehydrogenase gene.

The DNA of the present invention can be prepared by general genetic engineering techniques. For example, the DNA encoding the peptide tag of the present invention and the DNA encoding the useful protein can be prepared by PCR or DNA sequencing.
It can be constructed by ligating using a ligase or the like.

The recombinant vector of the present invention may be any vector in which DNA encoding the tagged protein is inserted so as to be expressible in a host cell into which the vector is introduced. The vector is not particularly limited as long as it is replicable in the host cell, and examples thereof include plasmid DNA, viral DNA, etc. Furthermore, it is preferable that the vector contains a selection marker such as a drug resistance gene. Specific examples of plasmid vectors include pTrcHis2 vector, pUC119, pBR322, pBluescript II KS+, pYES2, pAUR123, pQE-Tri, pET, pGEM-3Z, pGEX, pMAL, pRI909, pRI910, pBI221, pBI121, pBI101, pIG121Hm, pTrc99A, pKK223, pA1-11, pXT1, pRc/CMV, pRc/RSV, pcDNA I/Neo, p3×FLAG-CMV-14, pCAT3, pcDNA3.1, and pCMV.

The promoter used in the vector can be selected appropriately depending on the host cell into which the vector is introduced. For example, when expressing in yeast, the GAL1 promoter, PGK1 promoter, TEF1 promoter, ADH1 promoter, TPI1 promoter, PYK1 promoter, etc. can be used. When expressing in plants, the cauliflower mosaic virus 35S promoter, rice actin promoter, maize ubiquitin promoter, lettuce ubiquitin promoter, etc. can be used. When expressing in Escherichia coli, the T7 promoter can be used, and when expressing in Brevibacillus, the P2 promoter and P22 promoter can be used. Inducible promoters are also acceptable, such as the IPTG-inducible promoters lac, tac, and trc, as well as the IAA-inducible trp, L-arabinose-inducible ara, tetracycline-inducible Pzt-1, high-temperature (42°C)-inducible P _L promoter, and the promoter of the cspA gene, a cold shock gene.
If necessary, a terminator sequence may also be included depending on the host cell.

The recombinant vector of the present invention can be prepared, for example, by cleaving a DNA construct with an appropriate restriction enzyme or by adding a restriction enzyme site by PCR, and then inserting the DNA construct into a restriction enzyme site or multicloning site of a vector.

The transformant of the present invention is characterized in that it has been transformed with the above DNA or a recombinant vector containing it. The host cell used for transformation may be either a eukaryotic cell or a prokaryotic cell.
As eukaryotic cells, yeast cells, mammalian cells, plant cells, insect cells, etc. are preferably used. Examples of yeast include Saccharomyces cerevisiae , Candida utilis , Schizosaccharomyces pombe , Pichia pastoris, Yarrowia lipolytica, Metschnikowia pulcherrima , etc. Furthermore, microorganisms such as Aspergillus can also be used. Examples of prokaryotic cells include Escherichia coli , Lactobacillus , Bacillus subtilis, etc.
), Brevibacillus , Agrobacterium tumefaciens , Corynebacterium, cyanobacteria, actinomycetes, etc. Plant cells include Lactuca and other bacteria from the Asteraceae and Solanaceae families.
Examples include cells of plants belonging to the Brassicaceae, Rosaceae, and Chenopodiaceae families.

The transformant used in the present invention can be prepared by introducing the recombinant vector of the present invention into a host cell using a general genetic engineering technique. For example, methods that can be used include electroporation (Tada et al., 1990, Theor. Appl. Genet., 80:475), protoplast method (Gene, 39, 281-286(1985)), polyethylene glycol method (Lazzeri et al., 1991, Theor. Appl. Genet., 81:437), introduction methods using Agrobacterium (Hood et al., 1993, Transgenic. Res., 2:218; Hiei et al., 1994 Plant J., 6:271), particle gun method (Sanford et al., 1987, J. Part. Sci. Tech., 5:27), and polycation method (Ohtsuki et al., FEBS Lett., 1998 May 29;428(3):235-40). The gene expression may be transient expression or stable expression in which the gene is integrated into a chromosome.

After introducing the recombinant vector of the present invention into a host cell, a transformant can be selected based on the phenotype of the selection marker. The selected transformant can then be cultured to produce the tagged protein. The culture medium and conditions can be appropriately selected depending on the species of the transformant.
Furthermore, when the host cells are plant cells, the selected plant cells can be cultured according to a conventional method to regenerate the plant body, and the tagged protein can be accumulated inside the plant cells or outside the cell membrane of the plant cells.

In addition, a protein to which the peptide tag of the present invention has been added can also be expressed by introducing the DNA of the present invention, RNA (mRNA) transcribed therefrom, or a recombinant vector of the present invention into a cell-free expression system.
The cell-free expression system is not particularly limited as long as it is an expression system equipped with a protein expression mechanism such as ribosomes, but may also be a cell extract such as an Escherichia coli-derived cell extract, a wheat germ-derived cell extract, a rabbit reticulocyte-derived cell extract, or an insect cell-derived cell extract, or a protein expression system in which factors such as ribosomes are reconstituted.

Proteins carrying the peptide tag of the present invention that have accumulated in the medium, cells, or cell-free expression systems can be separated and purified using methods well known to those skilled in the art. For example, they can be separated and purified using known appropriate methods such as salting out, ethanol precipitation, ultrafiltration, gel filtration chromatography, ion exchange column chromatography, affinity chromatography, medium- to high-pressure liquid chromatography, reversed-phase chromatography, and hydrophobic chromatography, or a combination of these.

The following describes examples of the present invention, but the present invention is not limited to these examples.

(1) Construction of various plasmids encoding tagged GFP2 protein or VHH antibody for E. coli cell-free expression system Plasmids for expressing fusion proteins in which various peptide tags (Table 1) were added to the N-terminus or C-terminus of GFP2 protein or VHH antibody in an E. coli cell-free expression system or E. coli (BL21) were constructed according to the procedure described below.
Artificial synthetic DNA encoding GFP2 protein (SEQ ID NO: 70) or artificial DNA encoding VHH antibody (SEQ ID NO: 120) was inserted into the EcoRV recognition site of pUC19 modified plasmid pUCFa (Fasmac) to create various plasmids, pUCFa-GFP2 (Plasmid 1) and pUCFa-AmylD9 (
Plasmid 2) was obtained.
As a plasmid for expression in E. coli and cell-free systems, pET28a (Invitrogen) having a T7 promoter was used (Plasmid 3).
Using the procedures shown in Figure 1 or Figure 2, plasmids were constructed to express fusion proteins in which various peptide tags were added to the N-terminus or C-terminus of various proteins in an E. coli cell-free expression system or E. coli (BL21), respectively.
Next, to add various peptide tags to the N- or C-terminus of various proteins, PCR was performed using the combinations of template plasmids, forward primers, and reverse primers shown in Tables 2, 3, and 4. A sequence homologous to Plasmid 3 was added to the 5' end of each primer. PCR was performed using KOD-PLUS-Ver.2 (Toyobo) with 2 pg/μl template plasmid, 0.3 μ
A 50-μl reaction mixture was prepared containing 0.1 μM forward primer, 0.3 μM reverse primer, 0.2 mM dNTPs, 1× _Buffer for KOD-Plus-Ver.2, 1.5 mM MgSO4, and 0.02 U/μl KOD-PLUS-Ver.2. The mixture was heated at 94°C for 5 minutes, followed by 30 cycles of heating at 98°C for 10 seconds, 60°C for 30 seconds, and 68°C for 40 seconds, and finally at 68°C for 5 minutes. The amplified fragment was purified using a QIAquick PCR Purification Kit (Qiagen).
Plasmid 3 was digested with NcoI and HindIII, separated by electrophoresis using 1.0% SeaKem GTG Agarose, and extracted from the gel using a QIAquick Gel Extraction Kit (Qiagen).
Approximately 50 ng of extracted plasmid 3 (1 μl), 1 μl of purified PCR product, and 1 μl were mixed and adjusted to a volume of 3 μl. The mixture was then mixed with 0.75 μl of 5x In-Fusion HD Enzyme Premix provided with the In-Fusion HD Cloning Kit (TaKaRa), and left to stand at 50°C for 15 minutes, then left to stand on ice for 5 minutes.
1 μl of the reaction mixture was mixed with 15 μl of competent DH5-α cells, left on ice for 30 minutes, heated at 42°C for 45 seconds, left on ice for 2 minutes, added with 200 μl of SOC, and shaken at 37°C and 200 rpm for 1 hour. Then, the entire shaken mixture was spread on 2xYT agar medium containing 100 mg/L kanamycin and incubated at 37°C.
The colonies were then cultured overnight in 200 mL of PBS containing 100 mg/L kanamycin to obtain transformed colonies.
The resulting mixture was transferred to 4 ml of ×YT liquid medium and cultured overnight with shaking at 37°C and 200 rpm. The gene expression plasmid was extracted, and after confirming its base sequence, it was used in a cell-free expression test in E. coli and for transformation of E. coli (BL21(DE3)) strain.

The nucleotide sequences encoding each peptide tag are shown in SEQ ID NOs: 47 to 66 and 123 to 133. The nucleotide sequences encoding the 3-amino acid peptide tags are as follows: However, the codons in the nucleotide sequences encoding each peptide tag can be changed as long as the encoded amino acid sequence is the same.
2SR#242 (INK) ATAAATAAA
2SR#243 (INE) ATAAATGAA
2SR#244 (RNK) CGTAATAAA
2SR#245 (RND) CGTAATGAT

(2) Expression of Various Tagged Proteins Using a Cell-Free Expression System. PUREfrex 1.0 (Gene Frontier, Inc.) was used as the cell-free expression system. Solution I included with the kit was thawed at room temperature and then placed on ice. Solution II and Solution III were also thawed on ice. Solutions I, II, and III were vortexed briefly and spun down in a tabletop centrifuge. 25 μl each of sterile distilled water was added to the thawed Solution II and Solution III, vortexed, spun down, and mixed with the thawed Solution I. This mixture was thoroughly mixed by vortexing. A predetermined amount of plasmid and sterile distilled water were dispensed into a sterile 1.5 μl Eppendorf tube, and 8 μl of the mixture of Solutions I–III was added. Mixed by pipetting to avoid foaming, and then spun down in a tabletop centrifuge. The reaction was then carried out in a water bath at 37°C for 4 hours to express the protein. After the reaction was completed, 10 μl of sterile distilled water was added to the reaction mixture, followed by 20 μl of 2x sample buffer (ATTO Corporation), mixing, and then heating in a boiling bath for 10 minutes to prepare a sample for SDS-PAGE.

(3) Transformation of E. coli for Protein Expression. A glycerol stock of E. coli BL21 (DE3) (Novagen) was inoculated into a sterile 14 ml polystyrene tube containing 3 ml SOB medium (20 g/l Bacto tryptone, 5 g/l Bacto yeast extract, 10 mM NaCl, _2.5 mM KCl, 10 mM MgSO4, 10 mM _MgCl2 ) and cultured overnight at 37°C with shaking at 200 rpm. 0.2 ml of the above preculture was inoculated into a sterile Erlenmeyer flask containing 100 ml SOB medium and cultured at 30°C with shaking at 200 rpm. When the turbidity at 600 nm (OD600) reached 0.4-0.6, the culture was stopped by cooling on ice for 10-30 minutes. The culture was transferred to a 50 ml conical tube and centrifuged at 2,500 × g for 10 minutes at 4°C. The supernatant was discarded, and the pellet was gently suspended in 15 ml of ice-cold TB (10 mM PIPES-KOH, pH 6.7, 15 mM CaCl ₂ , 0.25 M KCl, 55 mM MnCl ₂ ).
The mixture was centrifuged at 37°C for 10 minutes. The supernatant was discarded, and 10 ml of ice-cold TB was added to the pellet and gently suspended. 700 μl of DMSO was added, and the mixture was suspended on ice. 50 ml of the mixture was placed in a 1.5 ml Eppendorf tube.
The cells were frozen in liquid nitrogen and stored at -80°C until use.
The resulting competent cells were thawed on ice, and 1 ng of the peptide-tagged protein expression plasmid for E. coli prepared above was added. The mixture was then gently mixed and allowed to stand on ice for 30 minutes.
After heat shock at 37°C for 45 seconds, the tube was left to stand on ice for 5 minutes. 250 μl of SOC was added, and the tube was placed horizontally and shaken at 200 rpm at 37°C for 1 hour. 100 μl of the shaken mixture was spread on 2xYT agar medium containing 100 mg/L kanamycin and cultured overnight at 37°C to obtain transformed colonies.

(4) Protein induction culture of E. coli After transformation, a single colony was smeared on a plate medium (2xYT, 100 mg/L kanamycin) and cultured overnight in an incubator at 37°C. After culture, the cells were scraped off from the plate medium with a sterile disposable loop and inoculated into a 14 ml sterile polystyrene tube containing 2 ml of pre-culture medium (2xYT, 100 mg/L kanamycin). The cells were then cultured at 37°C, 200 rpm, and OD600.
Shaking culture was performed until the OD600 value reached 0.6-1.0. The supernatants of these cultures were removed, and the resulting precipitates were treated with 1.0 ml of 2xYT medium (100 mg/l kanamycin). The amount of culture required to reach an OD600 value of 0.3 was transferred to a 1.5 ml Eppendorf tube and stored overnight at 4°C (in a refrigerator). The next day, the samples were centrifuged at 2,000 rpm for 30 min at 4°C. The supernatant was removed, and 1 ml of fresh 2xYT medium (100 mg/l kanamycin) was added to suspend the precipitate. Furthermore, 300 μl of each sample was inoculated into 2.7 ml of 2xYT medium (100 mg/l kanamycin) to achieve an OD600 value of 0.03. The cultures were then shaken at 37°C and 200 rpm until the OD600 value reached 0.4-1.0. Next, 3 μl of 1M IPTG (inducer) (final concentration 1 mM) was added, and the mixture was cultured at 30°C and 200 rpm for 12 hours with shaking. After the culture was completed, the test tube containing the sample was cooled on ice for 5 minutes to stop the growth of E. coli. 200 μl of the culture medium was then dispensed into a new 1.5 ml Eppendorf tube and centrifuged at 5,000 rpm, 4°C, and 5 minutes. The supernatant was then removed, and the cells were frozen in liquid nitrogen and then stored at -80°C.
The mixture was stored frozen at ℃.

(5) Protein Extraction from E. coli To the frozen sample, 100 μl of sample buffer (EZ Apply, manufactured by ATTO) was added, and the mixture was stirred using a vortex mixer. The mixture was then heated in boiling water for 10 minutes to convert the sample to SDS.

(6) Western analysis: Purified GFP2 protein was used as a standard substance for protein quantification. A dilution series was prepared by repeatedly diluting it two-fold with 1x sample buffer (ATTO Corporation), and this was used as a standard.
Protein electrophoresis (SDS-PAGE) was performed using an electrophoresis cell (BIO RAD) and Criterion TGX-gel (BIO RAD). The electrophoresis buffer (Tris/Glycine/SDS Buffer, BIO RAD) was added to the electrophoresis cell, and 10 μl of the SDS-modified sample was applied to the well. Electrophoresis was performed at a constant voltage of 200 V for 40 minutes.
After electrophoresis, the gel was blotted with Transblot Turbo (BIO RAD) using a Transblot Transfer Pack (BIO RAD).
After blotting, the membrane was immersed in blocking solution (TBS system, pH 7.2, Nacalai Tesque) and shaken at room temperature for 1 hour or left to stand at 4°C for 16 hours. Then, it was washed three times in TBS-T (137 mM sodium chloride, 2.68 mM potassium chloride, 1% polyoxyethylene sorbitan monolaurate, 25 mM Tris-HCl, pH 7.4) at room temperature for 5 minutes each with shaking.
To detect green fluorescent protein (GFP2), antiserum Rabbit-monoclonal Anti-GFP antibody ab32146 (Abcam) was used, and to detect VHH antibody (AmylD9), antiserum Rabbit-monoclonal Anti-VHH antibody A01860 (GenScript) was used, diluted 6,000-fold with TBS-T. The membrane was immersed in the diluted solution and shaken at room temperature for 2 hours to allow the antigen-antibody reaction, and then washed three times in TBS-T with shaking for 5 minutes at room temperature.
The secondary antibody was Anti-Rabbit IgG, AP-linked Antibody #7054 diluted 3,000 times in TBS-T.
The membrane was immersed in this dilution solution and shaken at room temperature for 1 hour to allow for the antigen-antibody reaction, and then washed three times in TBS-T with shaking at room temperature for 5 minutes. The alkaline phosphatase color reaction was performed using a color development solution (0.1 M sodium chloride, 5 mM magnesium chloride, 0.33 mg/ml nitroblue tetrazolium, 0.33 mg/ml 5-bromo-4-chloro-3-
The membrane was immersed in indolyl phosphate, 0.1 M Tris-HCl, pH 9.5, and shaken at room temperature for 15 minutes. The membrane was then washed with distilled water and dried on a Kimtowel at room temperature.
The colored membrane was imaged at a resolution of 600 dpi using a scanner (PM-A900, Epson), and various proteins were quantified using image analysis software (CS Analyzer ver. 3.0, ATTO Corporation).

(7) Measurement of GFP protein fluorescence intensity
A 100 μl aliquot of the GFP protein-induced culture sample was dispensed into a 96-well microplate and diluted two-fold with sterile distilled water. The fluorescence intensity (λEm) at 510 nm was measured using a Spectra Max iD5 fluorescence microplate reader (Molecular Devices) at an excitation wavelength (λEx) of 395 nm. The OD value of the same sample was also measured at 600 nm to estimate the amount of E. coli growth. The fluorescence intensity per OD value of 1.0 was then calculated by dividing the fluorescence intensity by the OD value.

(8) Construction of E. coli-Yarrowia lipolytica shuttle vector
A plasmid consisting of ori1001 (GenBank: EU340887.1) and Centromere1.1 (GenBank: AF099207.1) required for plasmid replication in Yarrowia lipolytica, ColE1 ori required for plasmid replication in E. coli, a hygromycin resistance gene (HYG), a TEF promoter required for metabolic enzyme expression, a multicloning site, and a CYC1 terminator was synthesized by FASMAC Co., Ltd. to obtain pEYHG (plasmid 4) (Figure 3, SEQ ID NO: 122).

(9) Construction of gene expression plasmids encoding various tagged GFP2 proteins for Yarrowia lipolytica
As in (1), the artificially synthesized DNA (SEQ ID NO: 70) encoding the GFP2 protein was inserted into the EcoRV recognition site of the pUC19 modified plasmid pUCFa (FASMAC), to obtain plasmid 1 (pUCFa-GFP2), which was used as a template.
Specifically, to add various tags (Table 1) to the N-terminus of GFP2 protein, PCR was performed using the template plasmid DNA, forward primer, and reverse primer combinations shown in Table 5. The 5' end of each primer contained a sequence homologous to that of plasmid 4. The resulting amplified fragment was purified with a QIAquick PCR Purification Kit (QIAGEN) and then inserted into plasmid 4 (pEYHG) digested with NotI and HindIII using the In-Fusion HD Cloning Kit (TaKaRa) according to the procedure shown in Figure 4 to obtain an expression plasmid. The constructed plasmid was then introduced into competent cells DH5-α (Nippon Gene Co., Ltd.) for cloning. The plasmid was then extracted, the nucleotide sequence confirmed, and used to transform Yarrowia lipolytica.

(10) Transformation of Yarrowia lipolytica
Yarrowia lipolytica was cultured in 150 mL of YPD-Rich medium (2% yeast extract, 4% peptone, 4% D-glucose, 0.01% tryptophan, 0.002% adenine) in a 500 mL baffled Erlenmeyer flask at 28°C, 180 rpm, and 16-18 hours with shaking. After confirming that the turbidity (OD600) reached 16-24, 400 μl of the culture was transferred to a sterile 1.5 mL Eppendorf tube and centrifuged at 500 g for 5 minutes at 4°C. The supernatant was removed, and 400 μl of 1 M sorbitol was added to the precipitate, which was then suspended and centrifuged again. After removing the supernatant, 400 μl of 1 M sorbitol was added to the precipitate, and the cells were suspended and centrifuged again. After removing the supernatant, 400 μl of 1 M sorbitol was added to the precipitate, and 1,000 ng of each of the plasmid DNAs constructed for transformation was added, followed by mixing using a vortex mixer.
200 μl of the above suspension was placed in a 0.2 cm cuvette for electroporation (Bio-Rad Laboratories, Gene
The resulting suspension was dispensed into a micropulser cuvette and electroporated twice per sample using a MicroPulser (Bio-Rad) at a voltage of 3.0 kV. 1,200 μl of YPD-Rich medium was added to 200 μl of the sample suspension and the mixture was shaken at 200 rpm at 28°C for 1 hour. After shaking, the mixture was centrifuged and the supernatant was removed. 1 ml of 1 M sorbitol was added to the precipitate to suspend it. 200 μl of the suspension was plated onto a YPDm plate (0.2% yeast extract, 5% peptone, 0.1% D-glucose, 50 mM sodium phosphate buffer pH 6.8, 2% agar). Transformant colonies were obtained after 5–7 days of static culture at 28°C.

(11) Cultivation and Sampling of Yarrowia lipolytica Clones in which the introduction of the target gene was confirmed by colony PCR were inoculated into 4 ml of YPD medium (1% peptone, 1% yeast extract, 6% glucose) in a 15 ml sterile round tube at an OD600 of 0.1, and cultured at 28°C and 200 rpm for 48 hours with shaking.
After the incubation, 100 μl of the culture was dispensed into a 1.5 ml Eppendorf tube and centrifuged at 4° C., 10,000 g for 5 minutes. The supernatant was removed, and the precipitate was used as a sample for Western analysis of the GFP2 protein.

(12) Enzyme extraction from Yarrowia lipolytica
GFP2 protein was extracted according to the method of Akira Hosomi et al. (Akira Hosomi, et al.: J Biol Chem, 285, (32), 24324-24334, 2010), by adding 100 μl of 0.1% HCl to the sample collected in (11).
N NaOH solution was added, the cells were suspended using a vortex mixer, and then the mixture was left to stand on ice for 10 minutes, then centrifuged at 4°C and 15,000 g for 5 minutes, the supernatant was discarded, and the precipitate was collected.

(13) Western analysis: 100 μl of sample buffer (EZ Apply, ATTO) was added to the resulting GFP2 protein precipitate, and the mixture was stirred using a vortex mixer. The mixture was then heated in boiling water for 10 minutes to desulfurize the sample. Subsequently, electrophoresis (SDS-PAGE) and blotting were performed using purified GFP as a standard in the same manner as in (6).
After blotting, the membrane was immersed in blocking solution (TBS system, pH 7.2, Nacalai Tesque) in the same manner as in (6), shaken at room temperature for 1 hour, and then resuspended in TBS-T (137 mM sodium chloride, 2.68 mM
The membranes were washed three times with shaking for 5 minutes at room temperature in a buffer containing potassium chloride, 1% polyoxyethylenesorbitan monolaurate, and 25 mM Tris-HCl, pH 7.4. To detect GFP2 protein, rabbit monoclonal anti-GFP antibody ab32146 (Abcam) was diluted 6,000-fold with TBS-T. The membrane was immersed in this diluted solution and shaken at room temperature for 2 hours to allow for the antigen-antibody reaction. The membrane was then washed three times with shaking for 5 minutes at room temperature in TBS-T. The secondary antibody used was anti-rabbit IgG, AP-linked antibody #7054 (Cell Signaling). The stained membranes were scanned at 600 dpi using a scanner (PM-A900, Epson), and the expression levels of various enzymes were measured using image analysis software (CS Analyzer ver. 3.0, ATTO).

(9) Results The results are shown in Figures 5 to 12.
As shown in Figure 5, in the cell-free expression system, the expression levels of the fusion proteins in which the peptide tags of Examples 1, 2, 4, 5, and 7 to 20 were linked to GFP2 were significantly improved compared to the fusion proteins in which the peptide tag described in Patent Document 4 (Comparative Example B) and the peptide tag (SKIK: SEQ ID NO: 22) described in Patent Document 3 (Comparative Example C) were linked to GFP2.

As shown in Figures 6 and 7, in the E. coli expression system, the expression levels of the fusion proteins in which the peptide tags of Examples 1 to 24 and 29 to 35 were linked to GFP2 were significantly improved compared to the fusion protein in which the peptide tag of Comparative Example B was linked to GFP2.
Furthermore, as shown in Figures 8 and 9, the fluorescence intensity of GFP2 was significantly higher in the fusion proteins in which the peptide tags of Examples 1 to 24 and 29 to 35 were linked to GFP2, confirming that functional proteins were expressed.

As shown in Figure 10, in the E. coli expression system, the expression levels of the fusion proteins in which the peptide tags of Examples 10, 12, and 15 were linked to the N-terminus of the VHH antibody were significantly improved compared to the fusion proteins in which the peptide tags of Comparative Examples B and D were linked to the N-terminus of the VHH antibody.
Furthermore, as shown in Figure 11, in the E. coli expression system, the expression levels of the fusion proteins in which the peptide tags of Examples 23, 29, and 31 were linked to the C-terminus of the VHH antibody were significantly improved compared to the fusion proteins in which the peptide tags of Comparative Examples B and D were linked to the C-terminus of the VHH antibody.

As shown in FIG. 12, in the Yarrowia lipolytica expression system,
The expression level of the fusion proteins in which peptide tags 18, 20, and 25 to 28 were linked to the N-terminus of GFP2 was significantly improved compared to the fusion proteins in which the peptide tags of Comparative Examples B and D and Comparative Example C described in Patent Document 3 were linked to the N-terminus of GFP2.

The peptide tags of the present invention are useful in fields such as genetic engineering and protein engineering, and proteins to which the peptide tags of the present invention have been added are useful in fields such as medicine, research, food, and livestock farming.

Claims (8)

A peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 7, 9 to 20 and SEQ ID NOs: 71 to 81, or any one of the amino acid sequences of INK, INE, RNK, and RND. A tagged protein comprising the peptide of claim 1 and a useful protein. The tagged protein of claim 2 , wherein the useful protein is an enzyme, cytokine, antibody, or fluorescent protein. A DNA encoding the tagged protein according to claim 2 or 3 . A recombinant vector comprising the DNA of claim 4 . A transformant transformed with the DNA of claim 4 or the recombinant vector of claim 5 . A method for producing a tagged protein, comprising culturing the transformant according to claim 6 to express and accumulate the tagged protein, and recovering the tagged protein. A method for producing a tagged protein, comprising introducing the DNA according to claim 4 or RNA transcribed therefrom into a cell-free expression system to express and accumulate the tagged protein, and recovering the tagged protein.