Peptide tags and tagged proteins containing the same

Abstract

To provide a peptide tag for improving an expression level of protein.SOLUTION: A peptide has an amino acid sequence shown in the following formulas (1), (2) or (3), and has the length of 3 to 6 amino acid. XmZnUq...(1), XmZnUqZn...(2), XZUZUZ...(3). Herein, X denotes amino acid residue independently selected from isoleucine, phenylalanine, methionine, alanine, valine, arginine, glutamine and glutamic acid, Z denotes amino acid residue independently selected from lysine and asparagine, U denotes amino acid residue independently selected from glycine, isoleucine, glutamine, valine, histidine, leucine, alanine, aspartic acid, glutamic acid, arginine and threonine, m and n denote an integer of 1 or 2, and q denotes an integer of 0, 1, 2 or 3.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] 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. [Background technology]

[0002] Thanks to advances in recombinant DNA technology, the production of useful proteins by heterologous expression is now commonplace. In the production of useful proteins by heterologous expression, various strategies for improving protein expression and accumulation have been investigated, 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).

[0003] In addition, several other techniques have been developed to improve expression of a target protein by linking a peptide tag to the protein (Patent Documents 2 to 6, Non-Patent Documents 1 to 3). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 5360727 specification [Patent Document 2] Patent No. 5273438 specification [Patent Document 3] International Publication No. WO2016 / 204198 Brochure [Patent Document 4] International Publication No. WO2017 / 115853 Brochure [Patent Document 5] International Publication No. WO2020 / 045530 Brochure [Patent Document 6] U.S. Patent Publication No. 20090137004 [Non-patent literature]

[0005] [Non-Patent Document 1] Smith, DB and Johnson, KS,: Gene, 67, 31, 1988 [Non-patent document 2] Marblestone, JG et al.: Protein Sci., 15, 182, 2006 [Non-patent document 3] di Guan, C. et al.: Gene, 67, 21, 1988 Summary of the Invention [Problem to be solved by the invention]

[0006] Patent Document 1 discloses that linking a toxin protein using a peptide linker in which prolines are spaced at regular intervals enables high accumulation of a toxin fusion protein in a plant. 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 premise of the presence of prolines spaced at regular intervals, and there is room for further study of the sequence to improve their performance as high-protein expression tags. Therefore, an objective of the present invention is to provide a novel peptide tag that can increase the expression level of a target protein by binding to the target protein when the target protein is expressed in a host cell or a cell-free expression system. [Means for solving the problem]

[0007] The present inventors investigated sequences to improve the performance of peptide tags. They then used a peptide tag having the amino acid sequence shown in formula (1) below and investigated the expression level of a protein to which this tag was attached. They found that the expression level of the target protein was significantly improved. The present invention was made based on this finding.

[0008] 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 , INE, RNK, or RND. peptides. [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] DNA encoding the tagged protein according to [5] or [6]. [8] A recombinant vector comprising the DNA described in [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. [Effects of the Invention]

[0009] Use of the peptide tag of the present invention can improve the expression level of a target protein, and is therefore useful for producing proteins using host cells such as yeast, Escherichia coli, and Brevibacillus, or cell-free expression systems. [Brief explanation of the drawings]

[0010]

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Figure 12

[0011] 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)

[0012] 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. X m 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.

[0013] Z is K or N, and m is 1 or 2, preferably 1. Z n means one or two consecutive Zs, and is one of the following: K N KK NN KN NK In equation (2), Z n There are two such cases, but they can be different, including the number of n. For example, X m NU q K, X m NU q N, X m NU q KN, X m NU q K.K., X. m NU q N.N., X m KU q K, X m KU q N, X mKU q KN, X m KU q K.K., X. m KU q Examples include NN. In formula (3), there are three Z's, which may be different from one another. Examples include XKUKUK, XKUKUN, XKUNUN, XNUNUN, XNUNUK, and XNUKUK.

[0014] 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 that q consecutive Us are present, and in this case, the q Us 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.

[0015] The peptide of the present invention has a length of 3 to 6 amino acids, more preferably 3 to 5 amino acids, even more preferably 4 to 5 amino acids, and particularly preferably 4 amino acids.

[0016] Specific examples of the peptides of the present invention are not particularly limited, but include, for example, 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.

[0017] The tagged protein of the present invention is a target protein to which the peptide tag of the present invention is bound (also referred to as a fusion protein of the tag and the target protein). A peptide tag may be attached to the end of the target protein, or the peptide tag may be attached to the C-terminus of the target protein. Alternatively, a peptide tag may be attached to both the N-terminus and C-terminus of the target protein. The peptide tag may be attached directly to the N-terminus and / or C-terminus of the target protein, or may be attached via a sequence of one to several amino acids (for example, 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, but by using 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. Furthermore, the tagged protein of the present invention may be tagged with a His tag, an HN tag, or the like. It may also contain other tag sequences necessary for detection, purification, etc., such as a FLAG tag.

[0018] Useful proteins contained in the tagged protein 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.

[0019] Examples of enzymes include lipase, protease, steroid synthesis enzyme, kinase, phosphatase, xylanase, esterase, methylase, demethylase, oxidase, reductase, cellulase, aromatase, collagenase, transglutaminase, glycosidase, and chitinase.

[0020] 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 growth factor (G-CSF), and These include phage colony-stimulating factor (GM-CSF), platelet-derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin (TPO), fibroblast growth factor (FGF), and hepatocyte growth factor (HGF).

[0021] Examples of hormones include insulin, glucagon, somatostatin, growth hormone, parathyroid hormone, prolactin, leptin, and calcitonin.

[0022] Examples of cytokines include interleukins, interferons (IFNα, IFNβ, IFNγ), and tumor necrosis factors (TNF).

[0023] Examples of blood proteins include thrombin, serum albumin, factor VII, factor VIII, factor IX, factor X, and tissue plasminogen activator.

[0024] 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 (scFvs), sc(Fv)2, disulfide-linked Fvs (sdFvs), diabodies, and VHH antibodies.

[0025] The antigen protein used as a vaccine is not particularly limited as long as it can induce an immune response, and may be appropriately selected depending on the target of the expected immune response. Examples of antigen proteins include proteins derived from pathogenic bacteria and pathogenic viruses.

[0026] The tagged protein of the present invention may have a secretory signal peptide added thereto that functions in host cells 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. When a plant is used as the host, the secretory signal peptide may be an invertase secretory signal, a P3 secretory signal, an α-factor secretory signal, or the like. Plants belonging to the Rosaceae, Brassicaceae, and Asteraceae families, more preferably the Nicotiana genus Nicotiana ), Arabidopsis thaliana ( Arabidopsis ), Rubus genus ( Fragaria ), Lactuca genus (Lactuca ), preferably tobacco ( Nicotiana tabacum ), Arabidopsis ( Arabidopsis thaliana ), Dutch strawberry ( Fragaria × ananassa ),lettuce( Lactuca sativa ) and the like.

[0027] Furthermore, the tagged protein of the present invention may be added with a transport signal peptide such as an endoplasmic reticulum retention signal peptide or a vacuolar transport signal peptide in order to express it in a specific cellular compartment.

[0028] The tagged proteins of the present invention can be chemically synthesized or produced by genetic engineering, as described below.

[0029] 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 reading frame.

[0030] DNA encoding a useful protein can be obtained by, for example, general genetic manipulation based on a known base sequence. It can be obtained by genetic engineering techniques. Furthermore, the DNA encoding the tagged protein of the present invention can be used to generate a host cell that produces the protein. It is also preferable that the codons representing the amino acids constituting the tagged protein are appropriately modified depending on the host cell so as to increase the translation yield of the hybrid protein. 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.

[0031] The DNA of the present invention is a gene that functions in a host cell in order to improve expression in the host cell. The enhancer may include an enhancer sequence such as a Kozak sequence. An example is the 5'-untranslated region of an alcohol dehydrogenase gene derived from a plant.

[0032] The DNA of the present invention can be prepared by general genetic engineering techniques, for example, DNA encoding the peptide tag and DNA encoding the useful protein are synthesized by PCR or DNA sequencing. It can be constructed by ligating using a ligase or the like.

[0033] The recombinant vector of the present invention is a vector in which the DNA encoding the tagged protein is The vector may be inserted into a vector so as to be expressible in the host cell into which it is introduced. The vector is not particularly limited as long as it is replicable in the host cell, and examples thereof include plasmid DNA and viral DNA. It is also preferable that the vector contains a selection marker such as a drug resistance gene. Specific examples of the plasmid vector 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.

[0034] The promoter used in the vector can be appropriately selected 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 can be used. For expression in E. coli, the T7 promoter can be used, and for expression in Brevibacillus, the P2 promoter or P22 promoter can be used. The promoter may be an inducible promoter, for example, lac, tac, trc promoters that are inducible by IPTG, trp promoters that are inducible by IAA, L-arabinose promoters, etc. ara, which is inducible with tetracycline; Pzt-1, which is inducible with high temperature (42°C); L A promoter such as the promoter of the cspA gene, which is one of the cold shock genes, can be used. If necessary, a terminator sequence may also be included depending on the host cell.

[0035] 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 inserting the DNA construct into the restriction enzyme site or multicloning site of a vector.

[0036] The transformant of the present invention is a transformant that has been transformed with the DNA or a recombinant vector containing the DNA. The host cells used for transformation may be either eukaryotic or prokaryotic cells. As eukaryotic cells, yeast cells, mammalian cells, plant cells, insect cells, etc. are preferably used. Saccharomyces cerevisiae or Candida utilis or Schizosaccharomyces pombeor Pichia pastoris, Yarrowia lipolytica, Metschnikowia pulcherrima In addition, koji mold ( Aspergillus ) and other microorganisms can also be used. As an example, E. coli ( Escherichia coli ), lactic acid bacteria ( Lactobacillus ), Bacillus subtilis ( Bacillus ), Brevibacillus ( Brevibacillus ), Agrobacterium ( Agrobacterium tumefaciens ), Corynebacterium, cyanobacteria, actinomycetes, etc. Plant cells include Lactuca genus ( Lactuca ) and other members of the Asteraceae family, Solanaceae family Examples include cells of plants belonging to the Brassicaceae, Rosaceae, and Chenopodiaceae families.

[0037] 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, such as 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 method using Agrobacterium (Hood et al., 1993, Transgenic. Res., 2:218; Hiei et al., 1994, Plant J., 6:271), particle method, etc. Methods such as the Gunn method (Sanford, et al., 1987, J. Part. Sci.tech. 5:27) and the polycation method (Ohtsuki, et al., FEBS Lett. 1998 May 29;428(3):235-40.) can be used. The gene expression may be transient expression or stable expression in which the gene is integrated into a chromosome.

[0038] 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.

[0039] 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.

[0040] Proteins carrying the peptide tag of the present invention that have accumulated in a medium, cells, or a cell-free expression system can be separated and purified by methods well known to those skilled in the art, 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, hydrophobic chromatography, or other known appropriate methods, or a combination of these.

[0041] Examples of the present invention will be described below, but the present invention is not limited to these examples. [Example]

[0042] (1) Construction of various plasmids encoding tagged GFP2 protein or VHH antibodies for E. coli cell-free expression systems Using the procedures described below, plasmids were constructed to express 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). Artificial synthetic DNA encoding GFP2 protein (SEQ ID NO: 70) or VHH antibody was used. The artificial DNA (SEQ ID NO: 120) was inserted into the EcoRV recognition site of the pUC19 modified plasmid pUCFa (Fasmac) to create various plasmids, pUCFa-GFP2 (plasmid 1) and pUCFa-AmylD9 ( Plasmid 2) was obtained. pET28a (Invitrogen) having a T7 promoter was used as a plasmid for expression in E. coli and cell-free systems (Plasmid 3). Using the procedures shown in Figure 1 or Figure 2, we constructed plasmids for expressing fusion proteins in an E. coli cell-free expression system or E. coli (BL21) in which various peptide tags were added to the N- or C-terminus of various proteins. 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 μl 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, followed by heating 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), left to stand at 50°C for 15 minutes, and then left to stand on ice for 5 minutes. 1 μl of the reaction mixture was mixed with 15 μl of competent cells DH5-α, and the mixture was left standing on ice for 30 minutes, then heated at 42°C for 45 seconds, and left standing on ice for 2 minutes. 200 μl of SOC was added, and the mixture was shaken at 37°C and 200 rpm for 1 hour. After that, the entire volume of the shaken mixture was spread on 2xYT agar medium containing 100 mg / l kanamycin, and then 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 cells were transferred to 4 ml of ×YT liquid medium and cultured overnight at 37°C with shaking at 200 rpm. After extracting and confirming the base sequence, a cell-free expression test was carried out in E. coli and E. coli (BL21(DE3)) strain. was used for transformation of

[0043] [Table 1] 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

[0044] [Table 2]

[0045] [Table 3]

[0046] [Table 4]

[0047] [Table 5]

[0048] (2) Expression of various tagged proteins using cell-free expression systems PUREfrex 1.0 (Gene Frontier Co., Ltd.) was used as the cell-free expression system. Solution I included in the kit was thawed at room temperature and then placed on ice. Solution II and Solution III included in the kit were placed on ice. Solution I, Solution II, and Solution III were vortexed briefly and then thawed on a tabletop centrifuge. After spinning down in a 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 mixed solution was mixed well with a vortex. A predetermined amount of plasmid and sterile distilled water were dispensed into a sterile 1.5 μl Eppendorf tube, and 8 μl of the mixed solution of Solutions I to III was added. Pipetting was performed without creating bubbles. The mixture was mixed equally and spun down in a tabletop centrifuge. Then, the mixture was placed in a water bath at 37°C for 4 hours. After the reaction was completed, 10 μl of sterile distilled water was added to the reaction mixture. After that, 20 μl of 2x sample buffer (ATTO Corporation) was added and mixed, and then the mixture was left in a boiling bath for 10 minutes. The mixture was heated for a short time and used as a sample for SDS-PAGE.

[0049] (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 of 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 the culture was shaken overnight at 37°C and 200 rpm. 0.2 ml of the above pre-culture solution was inoculated into a sterilized Erlenmeyer flask containing 100 ml of SOB medium, and cultured at 30°C with shaking at 200 rpm. When the turbidity at a wavelength of 600 nm (OD600) reached 0.4-0.6, the culture was incubated for 10-30 minutes. The culture was stopped by cooling on ice. The culture medium was transferred to a 50 ml conical tube and centrifuged at 2,500 × g at 4 °C for 10 minutes. 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 CaCl2, 0.25 M KCl, 55 mM MnCl2). The suspension was centrifuged at 2,500 × g at 4 °C for 10 minutes. 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. Ta. 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 4°C for 45 seconds, the cells were left on ice for 5 minutes. After adding 250 μl of SOC, The tube was then placed horizontally and shaken at 37°C and 200 rpm for 1 hour. 100 μl of the shaken mixture was added to 100 mg / L Kana. After spreading the culture on 2xYT agar medium containing .mycin, the culture was left to stand overnight at 37°C to obtain transformed colonies. Ta.

[0050] (4) Protein induction culture of E. coli After transformation, a single colony was plated onto a plate medium (2xYT, 100 mg / l kanamycin). The strain was strained and left to stand overnight in an incubator at 37°C. After the culture, the bacterial cells were scraped from the plate medium with a sterile disposable loop and transferred to 2 ml of pre-culture medium (2xYT, 100 mg / l Kanama). The cells were inoculated into a sterile polystyrene 14 ml tube containing 100 ml of PBS (Ishin), and incubated at 37°C, 200 rpm, and OD600 The culture was shaken until the OD600 value reached 0.6-1.0. When 1.0 ml of 2xYT medium (100 mg / l kanamycin) was added to the precipitate obtained by removing the supernatant from the culture, the OD600 value was 0.3. The required amount of culture was dispensed into a 1.5 ml Eppendorf tube and left to stand overnight at 4°C (in the refrigerator). The next day, the sample was centrifuged at 2,000 rpm at 4°C for 30 minutes, the supernatant was removed, and 1 ml of fresh 2xYT medium (100 mg / l kanamycin) was added to suspend the precipitate. Furthermore, 1 ml of the sample was added to 2.7 ml of 2xYT medium (100 mg / l kanamycin) so that the OD600 value was 0.03. 300 μl of the resulting solution was inoculated and cultured at 37°C with shaking at 200 rpm until the OD600 reached 0.4-1.0. Next, 3 μl of 1M IPTG (inducer) (final concentration 1 mM) was added, and cultured at 30°C with shaking at 200 rpm for 12 hours. After the incubation, the test tube containing the sample was cooled on ice for 5 minutes to screen the growth of E. coli. After the culture was allowed to top up, 200 μl of the culture medium was dispensed into a new 1.5 ml Eppendorf tube and centrifuged at 5,000 rpm at 4°C for 5 minutes. The supernatant was then removed, and the cells were frozen in liquid nitrogen and stored at -80°C. The mixture was stored frozen at ℃.

[0051] (5) Protein extraction from E. coli 100 μl of sample buffer (EZ Apply, manufactured by ATTO) was added to the frozen sample, 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.

[0052] (6) Western analysis The purified GFP2 protein was used as a standard substance for protein quantification. A dilution series was created by repeatedly diluting it two-fold with 1x sample buffer (ATTO Corporation). It was used as a standard. Protein electrophoresis (SDS-PAGE) was performed using an electrophoresis tank (Criterion cell, BIO RAD) and Criterion TGX-gel (BIO RAD) was used. An electrophoresis buffer (Tris / Glycine / SDS Buffer, BIO RAD) was placed in the electrophoresis tank, and 10 μl of the SDS-modified sample was applied to the wells. Electrophoresis was performed at a constant voltage of 200 V for 40 minutes. After electrophoresis, the gel was transblotted using a transblot transfer pack (BIO RAD). Blotting was performed using a blotting kit Turbo (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. The antiserum Rabbit-monoclonal Anti-GFP antibody ab32146 (Abcam) was used to detect green fluorescent protein (GFP2), and the antiserum Rabbit-monoclonal Anti-VHH antibody A01860 (GenScript) was used to detect VHH antibody (AmylD9), diluted 6,000-fold with TBS-T. The membrane was immersed in the solution and shaken at room temperature for 2 hours to allow the antigen-antibody reaction to occur. The plate was washed by shaking for 5 minutes three times. The secondary antibody was Anti-Rabbit IgG, AP-linked Antibody #7054 diluted 3,000 times in TBS-T. (Cell Signaling) was used. The membrane was immersed in this dilution solution and shaken at room temperature for 1 hour. The antigen-antibody reaction was carried out by this method, and the plate was washed three times in TBS-T by shaking at room temperature for 5 minutes. The color reaction using potassium phosphatase was carried out in a color development solution (0.1 M sodium chloride, 5 mM magnesium chloride). Nesium, 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).

[0053] (7) Fluorescence intensity measurement of GFP protein 100 μl of the GFP protein induction culture sample was dispensed into a 96-well microplate and sterilized. After diluting the sample two-fold with distilled water, the fluorescence intensity (λEm) at 510 nm was measured using a fluorescence microplate reader Spectra Max iD5 (Molecular Devices) with 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. was calculated.

[0054] (8) Construction of E. coli-Yarrowia lipolytica shuttle vector The ori1001 (GenBank: EU340887.1) and Centromere1.1 (GenBank: AF099207.1) for plasmid replication in Yarrowia lipolytica, the ColE1 ori for plasmid replication in E. coli, the hygromycin resistance gene (HYG), the TEF promoter for metabolic enzyme expression, and the multiclonal antibody A plasmid consisting of the CYC1 terminator and the CYC1 cloning site was synthesized by FASMAC Co., Ltd. to obtain pEYHG (plasmid 4) (Figure 3, SEQ ID NO: 122).

[0055] (9) Gene Expression Plus for Yarrowia lipolytica encoding various tagged GFP2 proteins Mido Construction As in (1), the artificially synthesized DNA encoding the GFP2 protein (SEQ ID NO: 70) was inserted into pUC19 modified Plasmid 1 (pUCFa-GFP2) obtained by inserting this gene into the EcoRV recognition site of mutant plasmid pUCFa (Fasmac) was used as a template. Specifically, in order to add various tags (Table 1) to the N-terminus of the GFP2 protein, the following methods were used: In addition, the combination of template plasmid DNA, forward primer, and reverse primer A sequence homologous to that of Plasmid 4 was added to the 5' end of each primer. The resulting amplified fragment was purified with a QIAquick PCR Purification Kit (QIAGEN) and then subjected to PCR amplification as shown in Figure 4. Following the procedure described above, the fragment was inserted into plasmid 4 (pEYHG) digested with Not I and Hind III using the In-Fusion HD Cloning Kit (TaKaRa) 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 base sequence was confirmed, and then used to transform Yarrowia lipolytica. Used.

[0056] (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 for 16 to 18 hours with shaking. Once the turbidity (OD600) reached 16 to 24, the culture was incubated. After confirmation, 400 μl of the culture was transferred to a sterile 1.5 ml Eppendorf tube and incubated at 4°C, 500 g, for 5 minutes. After centrifugation, the supernatant was removed, and 400 μl of 1 M sorbitol was added to the precipitate, which was then suspended and centrifuged again. 400 μl of 1 M sorbitol was added to the supernatant-removed precipitate, and the bacterial cells were suspended and centrifuged again. After further removal of the supernatant, 400 μl of 1 M sorbitol was added to the precipitate, and 1,000 ng of various plasmid DNAs constructed for transformation were added, and the mixture was mixed 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 suspension was dispensed into a Pulser Cuvette and electroporation was performed twice for each sample using a Micro Pulser (Bio-Rad) at a voltage of 3.0 kV. 200 μl of the sample suspension was added to 1,200 μl of YPD-Rich medium and shaken at 28°C and 200 rpm for 1 hour. After shaking, the suspension was centrifuged and the supernatant was removed. 1 ml of 1 M sorbitol was added to the precipitate to suspend it, and 200 μl of the suspension was plated on a YPDm plate medium (0.2% yeast extract, 5% peptone, 0.1% D-glucose, 50 mM sodium The cells were plated onto a 2% agar solution (2% agar, phosphate buffer, pH 6.8) and cultured at 28°C for 5 to 7 days to obtain transformed colonies.

[0057] (11) Cultivation and sampling of Yarrowia lipolytica The clones in which the introduction of the target gene was confirmed by colony PCR were cultured in YPD medium (1% peptone The cells were inoculated into a 15-ml sterile round tube containing 4 ml of the medium (1% yeast extract, 6% glucose) at an amount to give 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.

[0058] (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 the mixture was left to stand on ice for 10 minutes. Next, the mixture was centrifuged at 4°C and 15,000 g for 5 minutes, the supernatant was discarded, and the precipitate was collected.

[0059] (13) Western analysis To the resulting GFP2 protein precipitate, 100 μl of sample buffer (EZ Apply, manufactured by ATTO) was added, and after stirring with a vortex mixer, the sample was heated in boiling water for 10 minutes to convert it to SDS. The following was carried out by the same method as in (6), except that purified GFP was used as a standard for electrophoresis (SDS-PAGE) and blotting. After blotting, the membrane was washed with blocking solution (TBS, pH 7.2, NaCl) in the same manner as in (6). After immersion in TBS-T (137 mM sodium chloride, 2.68 mM sodium chloride, 2.68 mM sodium chloride), the plate was shaken at room temperature for 1 hour. The cells were washed three times with shaking for 5 minutes at room temperature in 25 mM Tris-HCl (pH 7.4, 1% polyoxyethylenesorbitan monolaurate, potassium chloride). GFP2 protein was detected using antiserum Rabbit-monoclonal Anti-GFP antibody ab32146 (Abcam) diluted 6,000-fold in TBS-T. The membrane was immersed in this dilution solution and shaken at room temperature for 2 hours to allow the antigen-antibody reaction to occur. The membranes were then washed three times in TBS-T with shaking for 5 minutes at room temperature. The secondary antibody used was Anti-Rabbit IgG, AP-linked Antibody #7054 (Cell Signaling). The stained membranes were imaged at a resolution of 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).

[0060] (9) Results The results are shown in Figures 5 to 12. As shown in FIG. 5, in a 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 higher than those of the peptide tag described in Patent Document 4 in Comparative Example B. In Comparative Example C, the peptide tag (SKIK: SEQ ID NO: 22) described in Patent Document 3 was linked to GFP2. This was a significant improvement compared to the fusion protein.

[0061] As shown in Figures 6 and 7, in the E. coli expression system, the results of Examples 1 to 24 and 29 to 35 were The expression level of the fusion protein in which a peptide tag was linked to GFP2 was significantly improved compared to the fusion protein in Comparative Example B in which a peptide tag was linked to GFP2. 8 and 9, the fluorescence intensity of GFP2 was significantly higher in the samples of Examples 1 to 24 and 29 to 35. The fusion protein in which the peptide tag was linked to GFP2 showed a significantly higher value, confirming that a functional protein was expressed.

[0062] 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.

[0063] As shown in FIG. 12, in the Yarrowia lipolytica expression system, Examples 10, 12, 15, Expression levels of fusion proteins in which peptide tags 18, 20, and 25-28 were linked to the N-terminus of GFP2 was significantly improved compared to the fusion protein 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. [Industrial Applicability]

[0064] The peptide tag of the present invention is useful in the fields of genetic engineering and protein engineering, and proteins to which the peptide tag of the present invention has been added are useful in the fields of medicine, research, food, livestock, and the like.

Claims

1. A peptide consisting of any of the amino acid sequences of SEQ ID NOs: 1 to 7, 9 to 20 and 71 to 81, or any of the amino acid sequences of INK, INE, RNK, and RND.

2. A tagged protein comprising the peptide of claim 1 and a useful protein.

3. The tagged protein of claim 2, wherein the useful protein is an enzyme, cytokine, antibody, or fluorescent protein.

4. A DNA encoding the tagged protein according to claim 2 or 3.

5. A recombinant vector comprising the DNA of claim 4.

6. A transformant transformed with the DNA of claim 4 or the recombinant vector of claim 5.

7. 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.

8. 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.

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