Peptide tags and tagged proteins containing the same

Abstract

Provided is a peptide having the following sequence and comprising 3-8 amino acid residues.　(I): XmZnPUq Here, P is proline; Z represents an amino acid residue independently selected from lysine (K) and asparagine (N); X represents an amino acid residue independently selected from isoleucine (I), phenylalanine (F), methionine (M), alanine (A), valine (V), tryptophan (W), tyrosine (Y), histidine (H), cysteine (C), arginine (R), glutamine (Q), and serine (S); and U represents an amino acid residue independently selected from arginine (R), glycine (G), serine (S), lysine (K), threonine (T), leucine (L), asparagine (N), histidine (H), and isoleucine (I).　m is 0, 1, 2, or 3; n is 1 or 2; and q is 0, 1, 2, or 3.

Description

[Technical Field]

[0001] The present invention relates to peptide tags and tagged proteins containing them, DNA encoding them, transformants containing the DNA, and methods for producing tagged proteins. [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 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 the sequence of peptide tags in order to improve their performance. They then used peptide tags with short amino acid sequences of 4 to 8 amino acid residues in which lysine (K) or asparagine (N) was placed before proline (P), and examined the expression level of proteins to which these were added. They found that the expression level of the target protein was significantly improved. The present invention was made based on these findings.

[0008] That is, the present invention is as follows. [1] A peptide having the following sequence and consisting of 3 to 8 amino acid residues: X m Z n PU q (I) where P is proline, Z is an amino acid residue independently selected from lysine (K) and asparagine (N); X is an amino acid residue independently selected from isoleucine (I), phenylalanine (F), methionine (M), alanine (A), valine (V), tryptophan (W), tyrosine (Y), histidine (H), cysteine ​​(C), arginine (R), glutamine (Q), and serine (S); U is an amino acid residue independently selected from arginine (R), glycine (G), serine (S), lysine (K), threonine (T), leucine (L), asparagine (N), histidine (H), and isoleucine (I). m is 0, 1, 2 or 3; n is 1 or 2; and q is 0, 1, 2 or 3. [2] The peptide according to [1], which consists of 3 to 6 amino acid residues. [3] The peptide according to [1] or [2], wherein m is 0 or 1 and q is 0 or 1. [4] The peptide according to any one of [1] to [3], having any one of the amino acid sequences of SEQ ID NOs: 1 to 18, SEQ ID NOs: 65 to 71, NKP, KNP, NNP, INP, MNP, QNP, IKP, HKP, SKP, MKP, and RKP. [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] [Figure 1] Schematic diagram of construction of tagged protein expression vector (tag added to the N-terminus). [Figure 2] Schematic diagram of construction of tagged protein expression vectors (tag added at the C-terminus) for E. coli and cell-free use. [Figure 3] Schematic diagram of the E. coli-Yarrowia lipolytica shuttle vector. [Figure 4] Schematic diagram of construction of tagged protein expression vector (tag added to the N-terminus) for Yarrowia lipolytica. [Figure 5]Schematic diagram of construction of tagged protein expression vector (tag added to the C-terminus) for Yarrowia lipolytica. [Figure 6] 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. [Figure 7] Graphs showing the expression level of tagged GFP2 in Escherichia coli (BL21) upon IPTG induction (Examples 1 to 6 and 8 to 18). The values ​​shown are relative to the expression level of untagged GFP2 (Comparative Example A), which is set to 1. [Figure 8] Graphs showing the fluorescence intensity of tagged GFP2 in Escherichia coli (BL21) upon IPTG induction (Examples 1 to 6, 8 to 18). The values ​​shown are relative to the fluorescence intensity of untagged GFP2 (Comparative Example A), which is set to 1. [Figure 9] Graph showing the expression level of tagged GFP2 in Escherichia coli (BL21) upon IPTG induction (Examples 19 to 36). The expression level is shown as a relative value when the expression level of untagged GFP2 (Comparative Example A) is set to 1. [Figure 10] Graphs showing the fluorescence intensity of tagged GFP2 in Escherichia coli (BL21) upon IPTG induction (Examples 19 to 36). The values ​​shown are relative to the fluorescence intensity of untagged GFP2 (Comparative Example A), which is set to 1. [Figure 11] 1 is a graph showing the expression level of N-terminally tagged VHH antibodies in Escherichia coli (BL21), where the expression level is relative to the expression level of untagged VHH antibodies (Comparative Example A), which is set to 1. [Figure 12] 1 is a graph showing the expression level of a VHH antibody tagged at its C-terminus in Escherichia coli (BL21), where the expression level is relative to the expression level of an untagged VHH antibody (Comparative Example A), which is set to 1. [Figure 13] Graph showing the expression level of GFP2 tagged at the N-terminus in Yarrowia lipolytica upon IPTG induction. The expression level of untagged GFP2 is shown as a relative value, with the expression level being set at 1. [Figure 14] Graph showing the expression level of GFP2 tagged at the C-terminus in Yarrowia lipolytica upon IPTG induction. The expression level of untagged GFP2 is shown as a relative value, with the expression level being set at 1. DETAILED DESCRIPTION OF THE INVENTION

[0011] The peptide of the present invention (also referred to as a peptide tag) has the following amino acid sequence: X m Z n PU q (I)

[0012] wherein P is proline, Z is an amino acid residue selected from lysine (K) and asparagine (N), and n is 1 or 2. Therefore, general formula (I) includes the following six types of sequences: X m KPU q (I-1) X m KNPU q (I-2) X m KKPU q (I-3) X m NPU q (I-4) X m NKPU q (I-5) X m NNPU q (I-6)

[0013] Each X is an amino acid residue independently selected from isoleucine (I), phenylalanine (F), methionine (M), alanine (A), valine (V), tryptophan (W), tyrosine (Y), histidine (H), cysteine ​​(C), arginine (R), glutamine (Q), and serine (S). X m means that m consecutive Xs are present, and in this case, the m Xs may be the same or different amino acid residues selected from I, F, M, A, V, W, Y, H, C, R, Q, and S. m is 0, 1, 2 or 3, preferably 0 or 1, and more preferably 1. In the above sequence (I-1) or (I-4), m is preferably not 0.

[0014] Each U is an amino acid residue independently selected from arginine (R), glycine (G), serine (S), lysine (K), threonine (T), leucine (L), asparagine (N), histidine (H), and isoleucine (I). For example, U is glycine (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 R, G, S, K, T, L, N, H, and I, or may be different amino acid residues. q is 0, 1, 2 or 3, preferably 0 or 1.

[0015] The peptide of the present invention has a length of 3 to 8 amino acids, more preferably 3 to 7 amino acids, and even more preferably 3 to 6 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 represented by SEQ ID NOs: 1 to 18, SEQ ID NOs: 65 to 71, NKP, KNP, NNP, INP, MNP, QNP, IKP, HKP, SKP, MKP, and RKP shown in Table 1 below.

[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 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.

[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 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. Furthermore, when a plant is used as the host, a plant belonging to the Solanaceae, Rosaceae, Brassicaceae, or Asteraceae family, more preferably a plant belonging to 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, 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.

[0031] 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.

[0032] The DNA of the present invention can be produced by general genetic engineering techniques, for example, by linking DNA encoding the peptide tag of the present invention and DNA encoding a useful protein using PCR, DNA ligase, etc.

[0033] 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.

[0034] 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. An inducible promoter may also be used, such as IPTG-inducible promoters lac, tac, and trc, as well as IAA-inducible trp, L-arabinose-inducible ara, tetracycline-inducible Pzt-1, and high-temperature (42°C)-inducible P. 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 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. Saccharomyces cerevisiae or Candida utilis or Schizosaccharomyces pombe or Pichia pastoris, Yarrowia lipolytica, Metschnikowia pulcherrima In addition, koji mold ( Aspergillus Microorganisms such as Escherichia 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 Examples include cells of plants belonging to the Asteraceae, Solanaceae, Brassicaceae, Rosaceae, and Chenopodiaceae families, such as Asteraceae, Solanaceae, Brassicaceae, and Chenopodiaceae.

[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. 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 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.

[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 the GFP2 protein (SEQ ID NO: 42) or artificial DNA encoding a VHH antibody (SEQ ID NO: 134) was inserted into the EcoRV recognition site of the pUC19 modified plasmid pUCFa (Fasmac) to obtain the plasmids pUCFa-GFP2 (Plasmid 1) and pUCFa-AmylD9 (Plasmid 2). pET28a (Invitrogen) having a T7 promoter was used as a plasmid for expression in E. coli and cell-free systems (Plasmid 3). Next, to add various peptide tags to the N- or C-terminus of the GFP2 protein or VHH antibody, PCR was performed using pUCFa-GFP2 (plasmid 1) or pUCFa-AmylD9 (plasmid 2) as a template and combinations of the template plasmids, forward primers, and reverse primers shown in Tables 2, 3, and 4. A sequence homologous to that of plasmid 3 was added to the 5' end of each primer. PCR was performed using KOD-PLUS Ver. 2 (Toyobo). A 50-μl reaction mixture was prepared containing 2 pg / μl template plasmid, 0.3 μM forward primer, 0.3 μM reverse primer, 0.2 mM dNTPs, 1× Buffer for KOD-Plus Ver. 2, 1.5 mM MgSO, and 0.02 U / μl KOD-PLUS Ver. 2. The mixture was heated at 94°C for 5 minutes, followed by 30 cycles of 98°C for 10 seconds, 60°C for 30 seconds, and 68°C for 40 seconds, followed by a final heating cycle 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 to adjust the volume to 3 μl, and then mixed with 0.75 μl of 5x In-Fusion HD Enzyme Premix provided with the In-Fusion HD Cloning Kit (TaKaRa). The mixture was left to stand at 50°C for 15 minutes and then on ice for 5 minutes. One μl of the reaction mixture was mixed with 15 μl of competent DH5-α cells and incubated on ice for 30 minutes, then heated at 42°C for 45 seconds. After incubation 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. The entire mixture was then plated onto 2xYT agar medium containing 100 mg / L kanamycin and incubated overnight at 37°C to obtain transformed colonies. The colonies were then transferred to 4 ml of 2xYT liquid medium containing 100 mg / L kanamycin and incubated overnight at 37°C and 200 rpm. The constructed plasmids for expression of tagged GFP2 protein or VHH antibodies were extracted using the procedures shown in Figures 1 and 2. The extracted plasmids were sequenced and used for cell-free expression tests in E. coli and for transformation of E. coli (BL21(DE3)).

[0043] [Table 1] The nucleotide sequences encoding each peptide tag (1 to 18 and Comparative Examples B and C) are shown in SEQ ID NOs: 21 to 40.

[0044] [Table 2]

[0045] [Table 3]

[0046] [Table 4]

[0047] [Table 5]

[0048] [Table 6]

[0049] (2) Expression of various peptide-tagged GFP2 proteins in a cell-free expression system PUREfrex 1.0 (Gene Frontier, Inc.) was used as a cell-free expression system. Solution I included with the kit was thawed at room temperature and then placed on ice. Solutions II and 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 Solutions II and III, vortexed, spun down, and mixed with the thawed Solution I. This mixture was thoroughly mixed by vortexing. A predetermined amount of the tagged GFP2 protein expression plasmid and sterile distilled water were placed in a sterile 1.5 μl Eppendorf tube, and 8 μl of the mixed solution of Solutions I–III was added. The mixture was mixed by pipetting, taking care not to create bubbles, 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.

[0050] (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 CaCl2, 0.25 M KCl, 55 mM MnCl2). The suspension was centrifuged at 2,500 x g at 4°C for 10 minutes. The supernatant was discarded, and the pellet was gently suspended in 10 ml of ice-cold TB. 700 μl of DMSO was added, and the mixture was suspended on ice. 50 μl aliquots were dispensed into 1.5 ml Eppendorf tubes to prepare competent cells. 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 above-prepared plasmid for expressing tagged GFP2 protein was added. The cells were then gently mixed and allowed to stand on ice for 30 minutes. After a 45-second heat shock at 42°C, the cells were allowed to stand on ice for 5 minutes. 250 μl of SOC was added, and the tube was placed horizontally and shaken at 37°C and 200 rpm for 1 hour. 100 μl of the shaken mixture was plated on 2xYT agar medium containing 100 mg / L kanamycin and incubated overnight at 37°C to obtain transformed colonies.

[0051] (4) Protein induction culture of E. coli After transformation, single colonies were plated onto plates (2xYT, 100 mg / L kanamycin) and incubated overnight in an incubator at 37°C. Bacteria were then scraped from the plates using a sterile disposable loop and inoculated into 14 ml sterile polystyrene tubes containing 2 ml of preculture medium (2xYT, 100 mg / L kanamycin). The cells were then cultured at 37°C and 200 rpm until an OD600 value of 0.6-1.0 was reached. The supernatants of these cultures were removed, and the amount of culture required to reach an OD600 value of 0.3 when 1.0 ml of 2xYT medium (100 mg / L kanamycin) was added to the precipitate was then transferred to a 1.5 ml Eppendorf tube and stored overnight at 4°C (in a 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, 300 μl of the sample was inoculated into 2.7 ml of 2xYT medium (100 mg / L kanamycin) to achieve an OD600 of 0.03. The culture was then cultured at 37°C and 200 rpm with shaking until the OD600 reached 0.4-1.0. Next, 3 μl of 1 M IPTG (inducer) (final concentration: 1 mM) was added, and the culture was cultured at 30°C and 200 rpm for 12 hours with shaking. After incubation, the test tube containing the sample was cooled on ice for 5 minutes to stop E. coli growth. Then, 200 μl of the culture was transferred to a new 1.5 ml Eppendorf tube and centrifuged at 5,000 rpm at 4°C for 5 minutes. Next, the supernatant was removed, and the cells were frozen in liquid nitrogen and then stored frozen at -80°C.

[0052] (5) Protein extraction from E. coli 100 μl of sample buffer (EZ Apply, ATTO Corporation) was added to the frozen sample, and after stirring with a vortex mixer, the sample was heated in boiling water for 10 minutes to convert it to SDS.

[0053] (6) Western analysis The standard substance used for protein quantification was a purified GFP2 protein preparation, which was diluted two-fold with 1x sample buffer (ATTO Corporation) to create a dilution series and used as the 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 with shaking for 5 minutes at room temperature in TBS-T (137 mM sodium chloride, 2.68 mM potassium chloride, 1% polyoxyethylene sorbitan monolaurate, 25 mM Tris-HCl, pH 7.4). 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 used was Anti-Rabbit IgG, AP-linked Antibody #7054 (Cell Signaling), diluted 3,000-fold with TBS-T. The membrane was immersed in this diluted solution and shaken at room temperature for 1 hour to allow for the antigen-antibody reaction. The membrane was then washed three times in TBS-T with shaking for 5 minutes at room temperature. The alkaline phosphatase color reaction was performed by immersing the membrane in 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-indolyl phosphate, 0.1 M Tris-HCl, pH 9.5) and shaking at room temperature for 15 minutes. The membrane was then washed with distilled water and dried on a Kimtowel at room temperature. The stained membrane was imaged at a resolution of 600 dpi using a scanner (PM-A900, Epson), and the GFP2 protein was quantified using image analysis software (CS Analyzer ver. 3.0, Atto Corporation).

[0054] (7) Fluorescence intensity measurement of GFP2 protein A 100 μl aliquot of the GFP2 protein induction culture sample was dispensed into a 96-well microplate and diluted 2-fold with sterile distilled water. The fluorescence intensity (λEm) at 510 nm was measured using a Spectra Max iD5 fluorescence microplate reader (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.

[0055] (8) Construction of E. coli-Yarrowia lipolytica shuttle vector A plasmid consisting of ori1001 (GenBank: EU340887.1) and Centromere1.1 (GenBank: AF099207.1) for plasmid replication in Yarrowia lipolytica, ColE1 ori for plasmid replication in E. coli, a hygromycin resistance gene (HYG), a TEF promoter 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: 136).

[0056] (9) Construction of gene expression plasmids encoding various tagged GFP2 proteins for Yarrowia lipolytica As in (1), the artificially synthesized DNA (SEQ ID NO: 42) 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- or C-terminus of the GFP2 protein, PCR was performed using the template plasmid DNA, forward primer, and reverse primer combinations shown in Tables 5 and 6. The 5' end of each primer contained a sequence homologous to that of plasmid 4. The resulting amplified fragment was purified using 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 procedures shown in Figures 4 and 5 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.

[0057] (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. Two hundred microliters of the suspension was dispensed into a 0.2 cm electroporation cuvette (Bio-Rad Gene Pulser Cuvette). Electroporation was performed twice per sample using a Micro Pulser (Bio-Rad) at a voltage of 3.0 kV. 1,200 μl of YPD-Rich medium was added to the 200 μl 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, and the precipitate was suspended. 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.

[0058] (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.

[0059] (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). 100 μl of 0.1 N NaOH solution was added to the sample collected in (11), the cells were suspended using a vortex mixer, and the mixture was left to stand on ice for 10 minutes. The mixture was then centrifuged at 4°C and 15,000 g for 5 minutes, the supernatant was discarded, and the precipitate was collected.

[0060] (13) Western analysis To the resulting GFP2 protein precipitate, 100 μl of sample buffer (EZ Apply, ATTO) was added, and the mixture was stirred using a vortex mixer. The sample was then heated in boiling water for 10 minutes to desulfurize the sample. Following this, 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, pH 7.2, Nacalai Tesque) and shaken at room temperature for 1 hour, followed by three 5-minute washes in TBS-T (137 mM sodium chloride, 2.68 mM potassium chloride, 1% polyoxyethylenesorbitan monolaurate, 25 mM Tris-HCl, pH 7.4) at room temperature. To detect GFP2 protein, rabbit monoclonal anti-GFP antibody ab32146 (Abcam) was diluted 6,000-fold in TBS-T. The membrane was immersed in this dilution and shaken at room temperature for 2 hours to induce antigen-antibody reactions. The membrane was then washed three times in TBS-T for 5 minutes each. Anti-Rabbit IgG, AP-linked Antibody #7054 (Cell Signaling) was used as the secondary antibody. The colored membrane was 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).

[0061] (8) Results The results are shown in Figures 6 to 14. As shown in Figure 6, in a cell-free expression system, the expression levels of the fusion proteins in which the peptide tags of Examples 3, 5, 7, and 9 to 18 were linked to the N-terminus of GFP2 were significantly improved compared to the fusion proteins in which the peptide tag described in Patent Document 4 of Comparative Example B and the peptide tag containing K but not P (SKIK: SEQ ID NO: 20 (Patent Document 3)) of Comparative Example C were linked to the N-terminus of GFP2.

[0062] As shown in Figures 7 and 9, in the E. coli expression system, the expression levels of the fusion proteins in which the peptide tags of Examples 1 to 6 and 8 to 36 were linked to the N-terminus of GFP2 were significantly improved compared to the fusion protein in which the peptide tag of Comparative Example B was linked to the N-terminus of GFP2. Furthermore, as shown in Figures 8 and 10, the fluorescence intensity of GFP2 was significantly higher in the fusion proteins in which the peptide tags of Examples 1 to 6 and 8 to 36 were linked to the N-terminus of GFP2, confirming that functional proteins were expressed.

[0063] 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 3, 9, 10, 15, 20, 21, 24, and 33 were linked to the N-terminus of the VHH antibody were significantly improved compared to the fusion proteins in Comparative Examples B and D in which the peptide tag described in Patent Document 4 was linked to the N-terminus of the VHH antibody.

[0064] As shown in Figure 12, in the E. coli expression system, the expression levels of the fusion proteins in which the peptide tags of Examples 9, 15, 33, and 35 were linked to the C-terminus of the VHH antibody were significantly improved compared to the fusion proteins in Comparative Examples B and D in which the peptide tag described in Patent Document 4 was linked to the C-terminus of the VHH antibody.

[0065] As shown in Figure 13, in the Yarrowia lipolytica expression system, the expression levels of the fusion proteins in which the peptide tags of Examples 3, 5, 9, 15, 16, 19 to 22, 24, 26, and 31 were linked to the N-terminus of GFP2 were significantly improved compared to the fusion proteins in which the peptide tags described in Patent Document 4 of Comparative Examples B and D and the peptide tag containing K but not P (SKIK: SEQ ID NO: 20 (Patent Document 3)) of Comparative Example C were linked to the N-terminus of GFP2.

[0066] As shown in Figure 14, in the Yarrowia lipolytica expression system, the expression levels of the fusion proteins in which the peptide tags of Examples 9, 15, 16, 19, and 35 were linked to the C-terminus of GFP2 were significantly improved compared to the fusion proteins in which the peptide tags described in Patent Document 4 of Comparative Examples B and D and the peptide tag containing K but not P (SKIK: SEQ ID NO: 20 (Patent Document 3)) of Comparative Example C were linked to the C-terminus of GFP2. [Industrial Applicability]

[0067] 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. Any of SEQ ID NOs: 1 to 12, 14 to 18, 65 to 71, KNP, INP, and HKP A peptide consisting of a sequence of amino acids.

2. The peptide according to claim 1 and a useful protein, and / or a tagged protein in which the peptide is directly bound as a tag to the C-terminus. 。

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 trait transformed with the DNA of claim 4 or the recombinant vector of claim 5. Transformation body.

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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