WO2022092132A1 - ペプチドタグおよびそれを含むタグ付加タンパク質 - Google Patents
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- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1024—Tetrapeptides with the first amino acid being heterocyclic
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Definitions
- the present invention relates to a peptide tag and a tagged protein containing the peptide tag, a DNA encoding the peptide tag, a transformant containing the DNA, and a method for producing the tagged protein.
- Patent Document 1 discloses a technique for expressing a bacterial toxin protein in a plant or the like, and discloses that the bacterial toxin protein is linked and expressed by a peptide linker in which proline is arranged at regular intervals (Patent). Document 1).
- Patent Documents 2 to 6 and Non-Patent Documents 1 to 3 Some techniques for improving the expression of the peptide tag by linking it to the target protein have been developed (Patent Documents 2 to 6 and Non-Patent Documents 1 to 3).
- Patent Document 1 By linking toxin proteins using a peptide linker in which proline disclosed in Patent Document 1 is arranged at regular intervals, it has become possible to highly accumulate toxin fusion proteins in plants. Further, in Patent Documents 4 and 5, amino acids between prolines in peptide tags were examined, and peptide tags suitable for high expression and soluble expression of proteins were provided. However, these peptide linkers and peptide tags are premised on the presence of prolines arranged at regular intervals, and there is room for further study of the sequences in order to improve the performance as a protein high expression tag. Therefore, it is an object of the present invention to provide a new peptide tag capable of increasing the expression level of the target protein by binding to the target protein when the target protein is expressed in a host cell or a cell-free expression system. And.
- the present inventors examined the sequence in order to improve the performance of the peptide tag. Then, using a peptide tag having a short amino acid sequence of 4 to 8 amino acid residues in which lysine (K) or asparagine (N) was arranged before proline (P), the expression level of the protein to which this was added was examined. However, it has been found that the expression level of the target protein is remarkably improved. The present invention has been made based on such findings.
- 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 isoleucine (I), phenylalanine (F), methionine (M), alanine (A), valine (V), tryptophan (W), tyrosine (Y), histidine (H), cysteine (C), arginine (R). ), Glutamine (Q), Serin (S), which is an amino acid residue independently selected.
- U is independent of arginine (R), glycine (G), serine (S), lysine (K), threonine (T), leucine (L), asparagine (N), histidine (H), isoleucine (I).
- R arginine
- G glycine
- S serine
- S lysine
- T threonine
- L leucine
- N histidine
- I isoleucine
- a method for producing a tagged-added protein which comprises culturing the transformant according to [9] to express and accumulate the tagged-added protein, and recovering the tagged-added protein.
- a tagged-added protein which comprises introducing the DNA according to [7] or RNA transcribed from the DNA into a cell-free expression system to express and accumulate the tagged-added protein, and recovering the tagged-added protein. Manufacturing method.
- the expression level of the target protein can be improved. Therefore, it is useful for protein production using host cells such as yeast, Escherichia coli, and Brevibacillus, and cell-free expression systems.
- FIG. 1 Schematic diagram of the construction of a tagged protein expression vector (tagged N-terminal addition).
- FIG. 1 Schematic diagram of construction of tag-added protein expression vector for Escherichia coli and cell-free (tag C-terminal addition).
- Schematic diagram of construction of tag-added protein expression vector for Yarrowia lipolytica (tag N-terminal addition).
- Schematic diagram of construction of tag-added protein expression vector for Yarrowia lipolytica (tag C-terminal addition).
- the graph which shows the expression level of the tagged green fluorescent protein (GFP2) in the cell-free expression system. The relative value when the expression level of untagged GFP2 (Comparative Example A) is 1 is shown.
- GFP2 green fluorescent protein
- Graphs showing the expression levels of tagged GFP2 in Escherichia coli (BL21) during IPTG induction (Examples 1 to 6, 8 to 18). The relative value when the expression level of untagged GFP2 (Comparative Example A) is 1 is shown.
- Graphs showing the fluorescence intensity of tagged GFP2 in Escherichia coli (BL21) during IPTG induction (Examples 1 to 6, 8 to 18).
- the relative value when the fluorescence intensity of untagged GFP2 (Comparative Example A) is 1 is shown.
- Graphs showing the expression level of tagged GFP2 in Escherichia coli (BL21) at the time of IPTG induction (Examples 19 to 36).
- the relative value when the expression level of untagged GFP2 (Comparative Example A) is 1 is shown.
- Graphs showing the fluorescence intensity of tagged GFP2 in Escherichia coli (BL21) during IPTG induction (Examples 19 to 36).
- the relative value when the fluorescence intensity of untagged GFP2 (Comparative Example A) is 1 is shown.
- the relative value when the expression level of the untagged VHH antibody (Comparative Example A) is 1 is shown.
- the relative value when the expression level of the untagged VHH antibody (Comparative Example A) is 1 is shown.
- the relative value when the expression level of untagged GFP2 is 1 is shown.
- the relative value when the expression level of untagged GFP2 is 1 is shown.
- the peptide of the present invention (also referred to as a peptide tag) has the following amino acid sequence.
- the 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)
- X is isoleucine (I), phenylalanine (F), methionine (M), alanine (A), valine (V), tryptophan (W), tyrosine (Y), histidine (H), cysteine (C), arginine ( It is an amino acid residue independently selected from R), glutamine (Q), and serine (S).
- X m means that m pieces of X are continuous, and in this case, m pieces of X are selected from I, F, M, A, V, W, Y, H, C, R, Q, and S. It may be the same amino acid residue or it may be a different amino acid residue.
- m is 0, 1, 2 or 3, preferably 0 or 1, and more preferably 1. In the sequence of (I-1) or (I-4), m is preferably not 0.
- U is independent of arginine (R), glycine (G), serine (S), lysine (K), threonine (T), leucine (L), asparagine (N), histidine (H), and isoleucine (I), respectively.
- I the amino acid residue selected.
- U is glycine (G).
- U q means that there are q consecutive U, and in this case, q U is the same amino acid residue selected from R, G, S, K, T, L, N, H, and I. It may be a different amino acid residue.
- q is 0, 1, 2 or 3, preferably 0 or 1.
- 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.
- peptide of the present invention are not particularly limited, but for example, SEQ ID NOs: 1 to 18, SEQ ID NOs: 65 to 71, NKP, KNP, NNP, INP, MNP, QNP, IKP, which are shown in Table 1 below. It is a peptide consisting of an amino acid sequence represented by any of HKP, SKP, MKP, and RKP.
- the tag-added protein of the present invention is a protein in which the peptide tag of the present invention is bound to the target protein (also referred to as a fusion protein of the tag and the target protein).
- the peptide tag may be bound to the N-terminal of the target protein, the peptide tag may be bound to the C-terminal of the target protein, or the peptide tag may be bound to both the N-terminal and the C-terminal of the target protein. good.
- the peptide tag may be directly attached to the N-terminal and / or C-terminal of the target protein, or may be attached via a sequence of 1 to several amino acids (for example, 1 to 5 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 added protein, but by using a protease recognition sequence, the peptide tag can be used as a useful protein after being expressed and purified. Can be separated from.
- the factor Xa recognition sequence is exemplified as the protease recognition sequence.
- the tagged added protein of the present invention may contain other tag sequences necessary for detection, purification, etc., such as His tag, HN tag, FLAG tag, and the like.
- the useful protein contained in the tagged protein of the present invention is not particularly limited, and examples thereof include growth factors, hormones, cytokines, blood proteins, enzymes, antigens, antibodies, transcription factors, receptors, fluorescent proteins or partial peptides thereof. Be done.
- Examples of the enzyme include lipase, protease, steroid synthase, kinase, phosphatase, xylanase, esterase, methylase, demethylase, oxidase, reductase, cellulase, aromatase, collagenase, transglutaminase, glycosidase and chitinase.
- Growth factors include, for example, epithelial growth factor (EGF), insulin-like growth factor (IGF), transforming growth factor (TGF), nerve growth factor (NGF), brain-derived neuronutrient factor (BDNF), and vascular endothelial cell proliferation.
- Factor (VEGF) vascular endothelial growth factor
- G-CSF Granulocyte Colony Stimulator
- GM-CSF Granulocyte Macrophage Colony Stimulator
- PDGF Thrombotic Growth Factor
- EPO Erythropoetin
- TPO Thrombopoetin
- FGF Fibroblast Growth Factor Factors
- HGF hepatocellular growth factor
- hormones include insulin, glucagon, somatostatin, growth hormone, parathyroid hormone, prolactin, leptin, and calcitonin.
- cytokines examples include interleukin, interferon (IFN ⁇ , IFN ⁇ , IFN ⁇ ), and tumor necrosis factor (TNF).
- IFN ⁇ interleukin
- IFN ⁇ interferon
- IFN ⁇ interferon
- TNF tumor necrosis factor
- blood proteins examples include thrombin, serum albumin, factor VII, factor VIII, factor IX, factor X, and tissue plasminogen activator.
- Antibodies include, for example, complete antibody, Fab, F (ab'), F (ab') 2 , Fc, Fc fusion protein, heavy chain (H chain), light chain (L chain), single chain Fv (scFv). , Sc (Fv) 2 , disulfide-bound Fv (sdFv), Diabody, VHH antibody.
- the antigen protein used as a vaccine is not particularly limited as long as it can elicit an immune response, and may be appropriately selected depending on the target of the assumed immune response.
- a protein derived from a pathogenic bacterium or pathogenicity may be selected. Examples include virus-derived proteins.
- the tagged-added protein of the present invention may be added with a secretory signal peptide that functions in a host cell for secretory production.
- Secreted signal Peptides include invertase secretory signal, P3 secretory signal, ⁇ -factor secretory signal, etc. when yeast is the host, PelB secretory signal when Escherichia coli is the host, and Brevibacillus as the host. In some cases, the P22 secretory signal is mentioned.
- plants belonging to the family Solanaceae, Rosaceae, Brassicaceae, and Asteraceae more preferably Nicotiana and Arabidopsis .
- Dutch strawberry Fragaria
- plants belonging to the genus Asteraceae Lactuca
- tobacco Nicotianatabacum
- rockcress Arabidopsis thaliana
- Dutch strawberry Fragaria ⁇ ananassa
- lettuce Lactuca sativa
- the tagged addition protein of the present invention may be added with a transport signal peptide such as an endoplasmic reticulum residual signal peptide and a vacuolar transfer signal peptide in order to express it in a specific cell compartment.
- a transport signal peptide such as an endoplasmic reticulum residual signal peptide and a vacuolar transfer signal peptide
- the tagged protein of the present invention can be chemically synthesized or genetically engineered. The method of producing by genetic engineering will be described later.
- the DNA of the present invention is characterized by containing a DNA encoding the tagged protein of the present invention. That is, the DNA of the present invention includes a DNA encoding a useful protein and a DNA encoding a peptide tag. The DNA encoding the useful protein and the DNA encoding the peptide tag are linked together in the reading frame.
- the DNA encoding a useful protein can be obtained, for example, by a general genetic engineering technique based on a known base sequence. Further, in the DNA encoding the tagged protein of the present invention, codons indicating amino acids constituting the tagged protein are appropriately modified so that the translation amount of the hybrid protein increases depending on the host cell producing the protein. It is also preferable to have. In addition, a method of selecting a codon that is frequently used in a host cell, a codon having a high GC content, or a codon that is frequently used in a housekeeping gene of a host cell can be mentioned.
- the DNA of the present invention may contain an enhancer sequence or the like that functions in the host cell in order to improve expression in the host cell.
- Enhancers include the Kozak sequence and the 5'-untranslated region of the plant-derived alcohol dehydrogenase gene.
- the DNA of the present invention can be produced by a general genetic engineering method.
- DNA encoding the peptide tag of the present invention, DNA encoding a useful protein, etc. are linked using PCR, DNA ligase, or the like. It can be built by doing.
- the recombinant vector of the present invention may be any as long as the DNA encoding the tagged protein is inserted into the vector so that it can be expressed in the host cell into which the vector is introduced.
- the vector is not particularly limited as long as it can be replicated in a host cell, and examples thereof include plasmid DNA and viral DNA.
- the vector preferably contains a selection marker such as a drug resistance gene.
- Specific plasmid vectors include, for example, pTrcHis2 vector, pUC119, pBR322, pBluescriptII KS +, pYES2, pAUR123, pQE-Tri, pET, pGEM-3Z, pGEX, pMAL, pRI909, pRI910, pBI221, pBI121, pBI101, pIG121Hm, Examples thereof include pTrc99A, pKK223, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, p3 ⁇ FLAG-CMV-14, pCAT3, pcDNA3.1, pCMV and the like.
- the promoter used in the vector can be appropriately selected depending on the host cell into which the vector is introduced.
- GAL1 promoter, PGK1 promoter, TEF1 promoter, ADH1 promoter, TPI1 promoter, PYK1 promoter and the like can be used.
- cauliflower mosaic virus 35S promoter, rice actin promoter, corn ubiquitin promoter, lettuce ubiquitin promoter and the like can be used.
- the T7 promoter and the like can be mentioned, and when expressed in brevibacillus, the P2 promoter and the P22 promoter can be mentioned.
- Pzt-1 a PL promoter that can be induced at high temperature (42 ° C)
- a promoter of the cspA gene which is one of the cold shock genes, and the like can be used.
- the terminator sequence can 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 adding a restriction enzyme site by PCR and inserting it into the restriction enzyme site or multicloning site of the vector.
- the transformant of the present invention is characterized by being transformed with the DNA or a recombinant vector containing the same.
- the host cell used for transformation may be either a eukaryotic cell or a prokaryotic cell.
- yeast cells yeast cells, mammalian cells, plant cells, insect cells and the like are preferably used.
- yeast include Saccharomyces cerevisiae , Candida utilis , Schizosaccharomyces pombe , Pichia pastoris, Yarrowia lipolytica, and Metschnikowia pulcherrima .
- microorganisms such as Aspergillus can also be used.
- proto-nuclear cells examples include Escherichia coli , Lactobacillus , Bacillus , Brevibacillus , Agrobacterium tumefaciens , corynebacterium, cyanobacteria, and actinomycetes.
- plant cells include cells of plants belonging to Asteraceae ( Astaraceae ), Brassicaceae (Brassicaceae), Rosaceae (Rosaceae), Asteraceae (Chenopodiaceae), etc. ..
- 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.
- a general genetic engineering technique For example, the electroporation method (Tada, et al., 1990, Theor. Appl. Genet, 80: 475), the protoplast method (Gene, 39, 281-286 (1985)), the 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.
- the gene expression may be transient expression or stable expression integrated into the chromosome.
- transformants After introducing the recombinant vector of the present invention into a host cell, transformants can be selected by the phenotype of the selectable marker.
- the tagged-added protein can be produced by culturing the selected transformant. The medium and conditions used for culturing can be appropriately selected depending on the species of the transformant.
- the host cell is a plant cell
- the plant can be regenerated by culturing the selected plant cell according to a conventional method, and the tagged addition protein is accumulated in the plant cell or outside the cell membrane of the plant cell. Can be made to.
- the DNA to which the peptide tag of the present invention is added can also be expressed by introducing the DNA of the present invention, RNA (mRNA) transcribed from the DNA, or the recombinant vector of the present invention into a cell-free expression system.
- RNA mRNA
- the cell-free expression system is not particularly limited as long as it is an expression system having a protein expression mechanism such as ribosome, but a cell extract derived from Escherichia coli, a cell extract derived from wheat germ, a cell extract derived from rabbit reticular erythrocytes, and insect cells. It may be a cell extract such as a cell extract of origin, or a protein expression system in which factors such as ribosomes are reconstituted.
- the protein to which the peptide tag of the present invention has been added accumulated in the medium, intracellular or cell-free expression system can be separated and purified according to a method well known to those skilled in the art.
- suitable methods such as salting out, ethanol precipitation, ultrafiltration, gel filtration chromatography, ion exchange column chromatography, affinity chromatography, medium and high pressure liquid chromatography, reverse phase chromatography, hydrophobic chromatography and the like. , Or a combination of these can be separated and purified.
- the artificial synthetic DNA encoding the GFP2 protein (SEQ ID NO: 42) or the artificial DNA encoding the VHH antibody (SEQ ID NO: 134) is inserted into the EcoRV recognition site of the pUC19 modified plasmid pUCFa (Fasmac) and the plasmid pUCFa-GFP2 (plasmid). 1) and pUCFa-AmylD9 (plasmid 2) were obtained.
- PET28a (Invitrogen) having a T7 promoter was used as a plasmid for E. coli and cell-free expression (plasmid 3).
- pUCFa-GFP2 (plasmid 1) or pUCFa-AmylD9 (plasmid 2) was used as a template to add various peptide tags to the N-terminus or C-terminus of the GFP2 protein or VHH antibody, and Tables 2, 3, and 4 PCR was performed using the combination of the template plasmid, forward primer, and reverse primer shown in. A sequence homologous to plasmid 3 was added to the 5'end of each primer.
- PCR uses KOD-PLUS-Ver.2 (Toyobo), 2 pg / ⁇ l template plasmid, 0.3 ⁇ M forward primer, 0.3 ⁇ M reverse primer, 0.2 mM dNTPs, 1 ⁇ Buffer for KOD-Plus-Ver.2, 1.5 mM.
- KOD-PLUS-Ver.2 Toyobo
- 2 pg / ⁇ l template plasmid 2 pg / ⁇ l template plasmid
- 0.3 ⁇ M forward primer 0.3 ⁇ M reverse primer
- 0.2 mM dNTPs 1 ⁇ Buffer for KOD-Plus-Ver.2, 1.5 mM.
- the obtained amplified fragment was purified with the 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 the QIAquick Gel Extraction Kit (Qiagen). Approximately 50 ng of extracted plasmid 3 1 ⁇ l, purified PCR product 1 ⁇ l and 1 ⁇ l are mixed to adjust the liquid volume to 3 ⁇ l, and then 5 ⁇ In-Fusion HD Enzyme Premix 0.75 attached to the In-Fusion HD Cloning Kit (TaKaRa). It was mixed with ⁇ l and allowed to stand at 50 ° C.
- the colonies were transplanted into 4 ml of 2 ⁇ YT liquid medium containing 100 mg / l kanamycin, cultured with shaking at 37 ° C. and 200 rpm overnight, and then tagged-added GFP2 constructed by the procedure shown in FIG. 1 or 2.
- a plasmid for expressing a protein or VHH antibody was extracted. After confirming the base sequence, the extracted plasmid was used for Escherichia coli cell-free expression test and transformation of Escherichia coli (BL21 (DE3)) strain.
- the cells were cultured with shaking at 30 ° C. and 200 rpm.
- the turbidity (OD600) at a wavelength of 600 nm reached 0.4-0.6, the cells were cooled on ice for 10-30 minutes and the culture was stopped.
- the culture was transferred to a 50 ml conical tube and centrifuged at 2,500 xg at 4 ° C for 10 minutes.
- the supernatant was discarded, and the pellet was added with 15 ml TB (10 mM PIPES-KOH, pH6.7, 15 mM CaCl 2 , 0.25 M KCl, 55 mM MnCl 2 ) ice-cooled and gently suspended.
- the suspension was centrifuged at 2,500 xg at 4 ° C for 10 minutes.
- the supernatant was discarded, and 10 ml TB of ice-cooled pellets were added and gently suspended.
- 700 ⁇ l of DMSO was added and suspended while cooling with ice. 50 ⁇ l each was dispensed into 1.5 ml Eppendorf tubes to make competent cells. After freezing in liquid nitrogen, it was stored at -80 ° C until use.
- the obtained competent cells were thawed on ice, 1 ng of the plasmid for expressing the tagged-added GFP2 protein prepared above was added, and the mixture was gently mixed and allowed to stand on ice for 30 minutes. After treatment at 42 ° C for 45 seconds (heat shock), the mixture was allowed to stand on ice for 5 min. After adding 250 ⁇ l of SOC, the tube was leveled and shaken at 37 ° C. and 200 rpm for 1 hour. 100 ⁇ l of the shaken product was applied to a 2 ⁇ YT agar medium containing 100 mg / l kanamycin, and then statically cultured at 37 ° C. overnight to obtain transformed colonies.
- the gel after electrophoresis was blotting with a transblot Turbo (BIO RAD) using a transblot transfer pack (BIO RAD).
- the membrane after blotting is immersed in a blocking solution (TBS system, pH7.2, Nakaraitesk), shaken at room temperature for 1 hour or allowed to stand at 4 ° C for 16 hours, and then TBS-T (137 mM sodium chloride, 2.68 mM potassium chloride, Washed in 1% polyoxyethylene sorbitan monolaurate, 25 mM Tris-HCl, pH 7.4) with 3 shakes at room temperature for 5 minutes.
- TBS system pH7.2, Nakaraitesk
- Antiserum Rabbit-monoclonal Anti-GFP antibody ab32146 (Abcam) for detection of green fluorescent protein (GFP2)
- antiserum Rabbit-monoclonal Anti-VHH antibody A01860 (GenScript) for detection of VHH antibody (AmylD9)
- TBS-T TBS-T
- the membrane was immersed in this diluted solution and shaken at room temperature for 1 hour to carry out an antigen-antibody reaction, and then shaken at room temperature for 5 minutes 3 times in TBS-T for washing.
- the color reaction by alkaline phosphatase is a color-developing 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-phosphoric acid, 0.1 M Tris.
- the membrane was immersed in HCl, pH 9.5,) and shaken at room temperature for 15 minutes.
- the membrane was washed with distilled water and then dried at room temperature on a Kim towel.
- the colored membrane was imaged with a scanner (PM-A900, Epson) at a resolution of 600 dpi, and GFP2 protein was quantified using image analysis software (CS Analyzer ver. 3.0, Atto Co., Ltd.).
- E. coli-Yarrowia lipolytica shuttle vector Ori1001 (GenBank: EU340887.1) and Centromere1.1 (GenBank: AF099207.1) for plasmid replication in Yarrowia lipolytica, ColE1 ori for plasmid replication in E. coli, hyglomycin resistance gene (HYG), metabolic enzyme
- a plasmid consisting of a TEF promoter, a multi-cloning site and a CYC1 terminator involved in expression was synthesized by Fasmac Co., Ltd. to obtain pEYHG (plasmid 4) (FIG. 3, SEQ ID NO: 136).
- a sequence homologous to plasmid 4 was added to the 5'end of each primer.
- the obtained amplified fragment was purified with the QIAquick PCR Purification Kit (QIAGEN), and then the In-Fusion HD Cloning Kit (PEYHG) digested with Not I and Hind III was added to the plasmid 4 (pEYHG) according to the procedure shown in FIGS. It was inserted using TaKaRa) to obtain an expression plasmid. Subsequently, the plasmid constructed in competent cell DH5- ⁇ (Nippon Gene Co., Ltd.) was introduced and cloned. Next, the plasmid was extracted, and after confirming the base sequence, it was used for transformation of Yarrowia lipolytica.
- the expression level of the fusion protein in which the peptide tags of Examples 1 to 6 and 8 to 36 were linked to the N-terminal of GFP2 was such that the peptide tag of Comparative Example B was the same as that of GFP2. It was significantly improved as compared with the fusion protein linked to the N-terminal. Further, as shown in FIGS. 8 and 10, the fluorescence intensity of GFP2 showed a remarkably high value in the fusion protein in which the peptide tags of Examples 1 to 6 and 8 to 36 were linked to the N-terminal of GFP2, and the functional protein showed a significantly high value. It was confirmed that it was expressed.
- the expression level of the fusion protein in which the peptide tags of Examples 9, 15, 16, 19, and 35 are linked to the C-terminal of GFP2 is the expression level of the fusion proteins of Comparative Examples B and D.
- the fusion protein in which the peptide tag described in 4 and the peptide tag of Comparative Example C containing K but not P (SKIK: SEQ ID NO: 20 (Patent Document 3)) linked to the C-terminal of GFP2 were significantly improved. ..
- the peptide tag of the present invention is useful in fields such as genetic engineering and protein engineering, and the protein to which the peptide tag of the present invention is added is useful in fields such as medicine, research, food, and livestock.
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Abstract
Description
[1]下記の配列を有し、3~8アミノ酸残基からなるペプチド。
XmZnPUq・・・(I)
ここで、Pはプロリンであり、
Zはリジン(K)およびアスパラギン(N)から独立して選択されるアミノ酸残基であり、
Xはイソロイシン(I)、フェニルアラニン(F)、メチオニン(M)、アラニン(A)、バリン(V)、トリプトファン(W)、チロシン(Y)、ヒスチジン(H)、システイン(C)、アルギニン(R)、グルタミン(Q)、セリン(S)から独立して選択されるアミノ酸残基であり、
Uはアルギニン(R)、グリシン(G)、セリン(S)、リジン(K)、トレオニン(T)、ロイシン(L)、アスパラギン(N)、ヒスチジン(H)、イソロイシン(I)から独立して選択されるアミノ酸残基である。
mは0、1、2または3であり、nは1または2であり、qは0、1、2または3である。
[2]3~6アミノ酸残基からなる、[1]に記載のペプチド。
[3]mが0または1であり、qが0または1である、[1]または[2]に記載のペプチド。
[4]配列番号1~18、配列番号65~71、NKP、KNP、NNP、INP、MNP、QNP、IKP、HKP、SKP、MKP、RKPのいずれかのアミノ酸配列を有する、[1]~[3]のいずれかに記載のペプチド。
[5][1]~[4]のいずれかに記載のペプチドと、有用タンパク質とを含む、タグ付加タンパク質。
[6]有用タンパク質が酵素、サイトカイン、抗体、または蛍光タンパク質である、[5]に記載のタグ付加タンパク質。
[7][5]または[6]に記載のタグ付加タンパク質をコードするDNA。
[8][7]に記載のDNAを含む組換えベクター。
[9][7]に記載のDNAまたは[8]に記載の組換えベクターで形質転換された形質転換体。
[10][9]に記載の形質転換体を培養してタグ付加タンパク質を発現及び蓄積させ、タグ付加タンパク質を回収することを特徴とする、タグ付加タンパク質の製造方法。
[11][7]に記載のDNAまたはそこから転写されるRNAを無細胞発現系に導入してタグ付加タンパク質を発現及び蓄積させ、タグ付加タンパク質を回収することを特徴とする、タグ付加タンパク質の製造方法。
XmZnPUq・・・(I)
したがって、一般式(I)には下記の6種類の配列が含まれる。
XmKPUq・・・(I-1)
XmKNPUq・・・(I-2)
XmKKPUq・・・(I-3)
XmNPUq・・・(I-4)
XmNKPUq・・・(I-5)
XmNNPUq・・・(I-6)
XmとはXがm個連続することを意味し、この場合のm個のXはI、F、M、A、V、W、Y、H、C、R、Q、Sから選択される同じアミノ酸残基であってもよいし、異なるアミノ酸残基であってもよい。
mは0、1、2または3であり、好ましくは0または1であり、より好ましくは1である。なお、上記(I-1)または(I-4)の配列においては、mは0ではないことが好ましい。
UqとはUがq個連続することを意味し、この場合のq個のUはR、G、S、K、T、L、N、H、Iから選択される同じアミノ酸残基であってもよいし、異なるアミノ酸残基であってもよい。
qは0、1、2または3であり、好ましくは0または1である。
また、本発明のタグ付加タンパク質をコードするDNAは、該タンパク質を生産させる宿主細胞に応じて、ハイブリッドタンパク質の翻訳量が増大するように、タグ付加タンパク質を構成するアミノ酸を示すコドンが適宜改変されていることも好ましい。また、宿主細胞において使用頻度の高いコドンを選択したり、GC含量が高いコドンを選択したり、宿主細胞のハウスキーピング遺伝子において使用頻度の高いコドンを選択したりする方法が挙げられる。
また、必要に応じ、ターミネーター配列も宿主細胞に応じて含めることができる。
真核細胞としては、酵母細胞、哺乳動物細胞、植物細胞、昆虫細胞などが好ましく用いられる。酵母としては、Saccharomyces cerevisiaeやCandida utilisやSchizosaccharomyces pombeやPichia pastorisやYarrowia lipolytica、Metschnikowia pulcherrimaなどが挙げられる。さらに、麹菌(Aspergillus)等の微生物を用いることもできる。原核細胞としては、大腸菌(Escherichia coli)、乳酸菌(Lactobacillus)、枯草菌(Bacillus)、ブレビバチルス(Brevibacillus)、アグロバクテリウム(Agrobacterium tumefaciens)、コリネバクテリウム、シアノバクテリア、放線菌などが挙げられる。植物細胞としては、アキノノゲシ属(Lactuca)などのキク科(Astaraceae)、ナス科(Solanaceae)、アブラナ科(Brassicaceae)、バラ科(Rosaceae)、アカザ科(Chenopodiaceae)に属する植物の細胞などが挙げられる。
また、宿主細胞が植物細胞の場合には、選抜した植物細胞を常法に従って培養することにより、植物体を再生することができ、植物細胞内または植物細胞の細胞膜外に前記タグ付加タンパク質を蓄積させることができる。
無細胞発現系はリボソームなどのタンパク質発現機構を備えた発現系であれば特に制限されないが、大腸菌由来の細胞抽出物、コムギ胚芽由来の細胞抽出物、ウサギ網状赤血球由来の細胞抽出物、昆虫細胞由来の細胞抽出物などの細胞抽出物や、リボソームなどの因子を再構成したタンパク質発現系でもよい。
下記手順で、GFP2タンパク質またはVHH抗体のN末端もしくはC末端に各種ペプチドタグ(表1)を付加した融合タンパク質を大腸菌無細胞発現系または大腸菌(BL21)で発現させるためのプラスミドをそれぞれ構築した。
GFP2タンパク質をコードする人工合成DNA(配列番号42)またはVHH抗体をコードする人工DNA (配列番号134)を、pUC19改変プラスミドpUCFa(ファスマック)のEcoRV認識部位に挿入してプラスミドpUCFa-GFP2(プラスミド1)およびpUCFa-AmylD9(プラスミド2)を得た。
大腸菌および無細胞系発現用プラスミドとしてT7プロモーターを有するpET28a(Invitrogen)を使用した(プラスミド3)。
次に、GFP2タンパク質またはVHH抗体のN末端もしくはC末端に各種ペプチドタグを付加するために、pUCFa-GFP2(プラスミド1)またはpUCFa-AmylD9(プラスミド2)を鋳型にし、表2、3、および4に示した鋳型プラスミド、フォワードプライマー、リバースプライマーの組合せによるPCRを実施した。各プライマーの5’末端にはプラスミド3との相同配列を付加した。PCRはKOD-PLUS-Ver.2(東洋紡)を用い、2 pg/μl 鋳型プラスミド、0.3 μM フォワードプライマー、0.3 μM リバースプライマー、0.2 mM dNTPs、1×Buffer for KOD-Plus-Ver.2、1.5 mM MgSO4、0.02 U/μl KOD-PLUS-Ver.2 となるように50 μl反応液を調製し、94℃、5分間加熱した後、98℃、10秒間、60℃、 30秒間、68℃, 40秒間の加熱処理を30サイクル行い、最後に68℃で5分間加熱した。得られた増幅断片はQIAquick PCR Purification Kit(Qiagen)で精製した。
プラスミド3はNcoI、HindIIIで消化後、1.0% SeaKem GTG Agaroseを用いた電気泳動により分離し、QIAquick Gel Extraction Kit(Qiagen)を用いゲルから抽出した。
約50 ng分の抽出したプラスミド3の1 μl、精製PCR産物1μlおよび1μlを混合し液量を3μlに調整後、In-Fusion HD Cloning Kit(TaKaRa)添付の5×In-Fusion HD Enzyme Premix 0.75μlと混合し、50℃で15分間静置、その後5分間氷上に静置した。
反応液1μlをコンピテントセルDH5-α 15μlと混合し氷上で30分間静置後、42℃で45秒間加温し、氷上で2分間静置後、SOCを200 μl添加し、37℃、200 rpmで1時間振盪した。その後、振盪物の全量を100 mg/lカナマイシンを含む2×YT寒天培地に塗布後、37℃で一晩静置培養し、形質転換コロニーを得た。次に、コロニーを100 mg/lカナマイシンを含む2×YT液体培地4 mlに移植し、37℃、200 rpmで一晩振盪培養した後、図1もしくは2に示した手順によって構築したタグ付加GFP2タンパク質またはVHH抗体発現用プラスミドの抽出を行った。抽出したプラスミドは、塩基配列を確認後、大腸菌無細胞発現試験および大腸菌(BL21(DE3))株の形質転換に用いた。
無細胞発現系としてPUREfrex 1.0(ジーンフロンティア株式会社)を用いた。Kitに添付のSolution Iを室温で融解後、氷上に静置した。添付のSolution II, Solution IIIは氷上で融解した。Solution I, Solution II, Solution IIIを軽くボルテックス後、卓上遠心機でスピンダウンした後、融解したSolution II, Solution IIIに滅菌蒸留水を各25μl加え、ボルテックス後、スピンダウンし、融解したSolution Iと混合した。この混合溶液をボルテックスでよく混ぜた。滅菌1.5μlエッペンドルフチューブに所定量のタグ付加GFP2タンパク質発現用プラスミドと滅菌蒸留水を分取し、更にSolution I~IIIの混合溶液8μl加え、ピペティングで泡立たない様に混合し、卓上遠心機でスピンダウンした。次に、37℃、4時間、ウォーターバス中で反応を行い、タンパク質を発現させた。反応終了後、反応物に10μlの滅菌蒸留水を加えた後、2xサンプルバッファー(アトー株式会社)を20μl加え混合後、沸騰浴中、10分間加熱し、SDS‐PAGE用サンプルとした。
大腸菌BL21 (DE3)(Novagen)のグリセロールストックを3 ml SOB培地(20 g/l Bacto tryptone、5 g/l Bacto Yeast Extract、10 mM NaCl、2.5 mM KCl、10 mM MgSO4、10 mM MgCl2)の入った滅菌ポリスチレン製14mlチューブに植菌し、37℃、200rpmで一晩振盪培養した。100 ml SOB培地の入った滅菌三角フラスコに上記前培養液を0.2 ml植菌後、30℃、200rpmで振盪培養した。波長600 nmでの濁度(OD600)が0.4-0.6になったら10-30分間氷冷し培養を止めた。培養液を50 ml コニカルチューブに移し2,500×g、4℃、10 分間遠心分離した。上清を捨て、ペレットを氷冷した15 ml TB(10 mM PIPES-KOH、pH6.7、15 mM CaCl2、0.25 M KCl、55 mM MnCl2)を加え穏やかに懸濁した。懸濁液を、2,500×g、4℃、10 分間遠心分離した。上清を捨て、ペレットに氷冷した10ml TBを加え穏やかに懸濁した。DMSOを700 μl加え、氷冷しながら懸濁した。1.5 mlエッペンドルフチューブに50μlずつ分注しコンピテントセルとした。液体窒素で凍結後、使用するまで‐80℃保存した。
得られたコンピテントセルを氷上で融解して、上記で作製したタグ付加GFP2タンパク質発現用プラスミドを1 ng添加後、穏やかに混合し氷上で30 分間静置した。42℃、45秒処理(ヒートショック)した後、氷上で5 min静置した。250 μlのSOCを添加後、チューブを水平にし、37℃、200 rpmで1 時間振盪した。振盪物100μlを100 mg/lカナマイシンを含む2×YT寒天培地に塗布後、37℃で一晩静置培養し、形質転換コロニーを得た。
形質転換後のシングルコロニーをプレート培地(2×YT、100mg/l カナマイシン)に塗株し、37℃、一晩インキュベーター中に静置し培養を行った。次に、培養後のプレート培地より滅菌ディスポループで菌体をかきとり、2 ml前培養培地(2×YT, 100mg/l カナマイシン)を分注した滅菌ポリスチレン製14 mlチューブに植菌し、37℃、200 rpm、OD600値が0.6~1.0に達するまで振盪培養を行った。これら培養物の遠心上清を除いた沈殿物に1.0ml 2×YT培地(100mg/l カナマイシン)を添加した際に、OD600値が0.3になるのに必要な量の培養物を、1.5ml エッペンドルフチューブに分取し、4℃(冷蔵庫内)で一晩静置保管した。翌日、前記サンプルを2,000 rpm、4℃、30 min遠心後、上清を除去し、新しい2×YT 培地(100mg/l カナマイシン)1 mlを加え、沈殿を懸濁した。更に、OD600値が0.03となる様に、2.7 mlの2×YT 培地(100mg/l カナマイシン)に上記サンプル1 mlのうちの300μlを植菌し、OD600値が0.4~1.0に達するまで、37℃、200 rpmで振盪培養した。 次に、1M IPTG(誘導剤)を3μl(終濃度1mM)加え、30℃、200 rpmで12時間振盪培養を行った。培養終了後、サンプルの入った試験管を氷上で5分間冷却し、大腸菌の増殖をストップさせた後、培養液200μlを新しい1.5 mlのエッペンドルフチューブに分取し、5,000rpm、4℃、5min遠心分離を行った。次に、上清を除き、菌体を液体窒素で凍結後、‐80℃で凍結保存した。
凍結保存サンプルに100μlのサンプルバッファー(EZ Apply、アトー株式会社)を加え、ボルテックスミキサーにて撹拌後、沸騰水中で10分間加熱し、サンプルのSDS化を行った。
タンパク質定量時の標準物質にはGFP2タンパク質精製標品を用いた。これを1xサンプルバッファー(アトー株式会社)で2倍希釈を繰り返すことにより希釈系列を作成し、スタンダードとして用いた。
タンパク質の電気泳動(SDS-PAGE)は、電気泳動槽(Criterion cell、BIO RAD)およびCriterion TGX-ゲル(BIO RAD)を用いた。電気泳動槽に泳動バッファー(Tris/Glycine/SDS Buffer、BIO RAD)を入れ、ウェルにSDS化したサンプルを10 μlアプライし、200 V定電圧で40分間泳動した。
電気泳動後のゲルは、トランスブロット転写パック(BIO RAD)を用い、トランスブロットTurbo(BIO RAD)でブロッティングを行った。
ブロッティング後のメンブレンはブロッキング溶液(TBS系, pH7.2、ナカライテスク)に浸し、室温で1時間振盪または4℃で16時間静置後、TBS-T(137 mM 塩化ナトリウム、2.68 mM 塩化カリウム、1% ポリオキシエチレンソルビタンモノラウラート、25 mM Tris-HCl、pH 7.4)中で室温、5分間の振盪を3回行い洗浄した。
緑色蛍光タンパク質(GFP2)の検出には、抗血清Rabbit-monoclonal Anti-GFP antibody ab32146 (アブカム)を、VHH抗体(AmylD9)の検出には、抗血清Rabbit-monoclonal Anti-VHH antibody A01860 (GenScript)をTBS-Tで6,000倍希釈して使用した。希釈液中にメンブレンを浸し、室温で2時間振盪することにより抗原抗体反応を行い、TBS-T中で室温、5分間の振盪を3回行い洗浄した。
二次抗体には、TBS-Tで3,000倍希釈したAnti-Rabbit IgG, AP-linked Antibody #7054(Cell Signaling)を使用した。本希釈液中にメンブレンを浸し、室温で1時間振盪することにより抗原抗体反応を行い、TBS-T中で室温、5分間の振盪を3回行い洗浄した。アルカリホスファターゼによる発色反応は、発色液(0.1 M 塩化ナトリウム、5 mM 塩化マグネシウム、0.33 mg/mlニトロブルーテトラゾリウム、0.33 mg/ml 5-ブロモ-4-クロロ-3-インドリル-リン酸、0.1 M Tris-HCl、pH9.5、)中にメンブレンを浸し、室温で15分間振盪することにより行い、メンブレンを蒸留水で洗浄した後、キムタオル上で常温乾燥した。
発色したメンブレンはスキャナー(PM-A900、エプソン)により解像度600 dpiで画像化し、画像解析ソフト(CS Analyzer ver. 3.0、アトー株式会社)を用い、GFP2タンパク質の定量を行った。
GFP2タンパク質の誘導培養サンプル100μlを96ウェルマイクロプレートに分取し、滅菌蒸留水で2倍希釈した後、蛍光マイクロプレートリーダーSpectra Max iD5(Molecular DEVICES)を用いて、励起波長(λEx)395 nmで、510 nmの蛍光強度(λEm)を測定した。併せて、同一のサンプルにつき、600 nmでOD値を測定し、大腸菌の増殖量を見積もった。次に、上記OD値で前記の蛍光強度を除することにより、OD値1.0当たりの蛍光強度を算出した。
Yarrowia lipolytica内でのプラスミド複製に係るori1001(GenBank:EU340887.1)およびCentromere1.1(GenBank:AF099207.1)、大腸菌内でのプラスミド複製に係るColE1 ori、ハイグロマイシン耐性遺伝子 (HYG)、代謝酵素発現に係るTEFプロモーター、マルチクローニングサイトおよびCYC1ターミネーターから成るプラスミドを株式会社ファスマックで合成し、pEYHG(プラスミド4)を取得した(図3、配列番号136)。
(1)と同様に、GFP2タンパク質をコードする人工合成DNA(配列番号42)を、pUC19改変プラスミドpUCFa(ファスマック)のEcoRV認識部位に挿入して得たプラスミド1(pUCFa-GFP2)を鋳型として用いた。
具体的には、GFP2タンパク質のN末端もしくはC末端に各種タグ(表1)を付加するために、表5および6に示した、鋳型プラスミドDNA、フォワードプライマー、リバースプライマーの組み合わせによるPCRを実施した。各プライマーの5’末端にはプラスミド4との相同配列を付加した。得られた増幅断片はQIAquick PCR Purification Kit(QIAGEN)で精製した後、図4および5に示した手順に従い、Not I、Hind IIIで消化したプラスミド4(pEYHG)に、In-Fusion HD Cloning Kit(TaKaRa)を用いて挿入し発現用プラスミドを得た。続いて、コンピテントセルDH5-α(株式会社ニッポンジーン)に構築したプラスミドを導入し、クローニングを行った。次に、プラスミドを抽出し、塩基配列を確認後、Yarrowia lipolyticaの形質転換に用いた。
Yarrowia lipolyticaを500mlのバッフル付き三角フラスコを用いて、YPD-Rich培地150 mL(2% yeast extract, 4% peptone, 4% D-glucose, 0.01% Tryptophan, 0.002% Adenine)で28℃、180rpm、16~18時間、振盪培養した。濁度(OD600)が16~24になったのを確認後、培養物400μlを滅菌した1.5mlエッペンドルフチューブに取り、4℃、500g、5分間遠心分離した後、上清を除去後、沈殿に1Mソルビトール400μlを加え、懸濁後、再び遠心分離を行った。上清を除去した沈殿に再び1Mソルビトール400μlを加え、菌体を懸濁後、遠心分離を行った。更に上清を除去した後、沈殿に1Mソルビトール400μlを加えるとともに、形質転換用に構築した各種プラスミドDNAを1,000ng加え、ボルテックスミキサーで混合した。
上記の懸濁液200μlをエレクトロポレーション用0.2 cmキュベット(Bio-Rad社製 Gene Pulser Cuvette)に分注し、Micro Pulser(Bio-Rad社製)を用いて、エレクトロポレーションを3.0 KVの電圧で、一つのサンプルにつき2回実施した。サンプル懸濁液200μlにYPD-Rich培地を1,200μl加え、28 ℃、200 rpmで1時間振盪した。振盪後、遠心分離を行い、上清を除去した後、沈殿に1 mlの1Mソルビトールを加え、沈殿を懸濁し、懸濁液200μlをYPDmプレート培地(0.2% yeast extract, 5% peptone, 0.1% D-glucose, 50mM ナトリウム-リン酸緩衝液 pH6.8, 2% Agar)に塗布した。28℃で5~7日静置培養し、形質転換コロニーを得た。
コロニーPCRにより目的遺伝子の導入の確認できたクローンを、YPD培地(1% ペプトン、1% 酵母エキス、6% グルコース)4 mlを分注した15 ml滅菌ラウンドチューブにOD600が0.1になる量を植菌し、28℃、200rpmで48時間振盪培養を行った。
培養後、培養物100μlを1.5mlエッペンドルフチューブに分取し、4℃、10,000g、5分間、遠心分離を行い、上清を除去した後、沈殿をGFP2タンパク質のウェスタン解析用サンプルとした。
GFP2タンパク質の抽出はAkira Hosomi らの方法(Akira Hosomi, et al,: J Biol Chem, 285, (32), 24324-24334, 2010)に従い、(11)でサンプリングした試料に100μlの0.1N NaOH溶液を加え、ボルテックスミキサーにて菌体を懸濁した後、氷冷化10分静置した。次に、4℃、15,000g、5分間遠心分離を行い、上清を棄てた後、沈殿を回収した。
得られたGFP2タンパク質の沈殿に100μlのサンプルバッファー(EZ Apply、ATTO製)を加え、ボルテックスミキサーにて撹拌後、沸騰水中で10分間加温し、サンプルのSDS化を行った。以下は、(6)と同様の方法によって、精製GFPを標品として電気泳動(SDS-PAGE)及び、ブロッティングを行った。
ブロッティング後も(6)と同様に、メンブレンをブロッキング溶液(TBS系, pH7.2、ナカライテスク)に浸し、室温で1時間振盪後、TBS-T(137 mM 塩化ナトリウム、2.68 mM 塩化カリウム、1% ポリオキシエチレンソルビタンモノラウラート、25 mM Tris-HCl、pH 7.4)中で室温、5分間の振盪を3回行い洗浄した。GFP2タンパク質の検出には、抗血清Rabbit-monoclonal Anti-GFP antibody ab32146 (アブカム)をTBS-Tで6,000倍希釈して使用した。本希釈液中にメンブレンを浸し、室温で2時間振盪することにより抗原抗体反応を行い、TBS-T中で室温、5分間の振盪を3回行い洗浄した。二次抗体には、Anti-Rabbit IgG, AP-linked Antibody #7054(Cell Signaling)を使用した。発色したメンブレンはスキャナー(PM-A900、エプソン)により解像度600 dpiで画像化し、画像解析ソフト(CS Analyzer ver. 3.0、アトー)を用い、各種酵素の発現量を測定した。
結果を図6~14に示す。
図6に示すように、無細胞発現系において、実施例3、5、7、9~18のペプチドタグをGFP2のN末端に連結した融合タンパク質の発現量は、比較例Bの特許文献4記載のペプチドタグや比較例CのKは含むがPは含まないペプチドタグ(SKIK:配列番号20(特許文献3))をGFP2のN末端に連結した融合タンパク質に比べて、顕著に向上した。
また、図8および10に示すように、GFP2の蛍光強度は実施例1~6、8~36のペプチドタグをGFP2のN末端に連結した融合タンパク質において顕著に高い値を示し、機能的タンパク質が発現されていることが確認できた。
Claims (11)
- 下記の配列を有し、3~8アミノ酸残基からなるペプチド。
XmZnPUq・・・(I)
ここで、Pはプロリンであり、
ZはK(リジン)およびアスパラギン(N)から独立して選択されるアミノ酸残基であり、
Xはイソロイシン(I)、フェニルアラニン(F)、メチオニン(M)、アラニン(A)、バリン(V)、トリプトファン(W)、チロシン(Y)、ヒスチジン(H)、システイン(C)、アルギニン(R)、グルタミン(Q)、セリン(S)から独立して選択されるアミノ酸残基であり、
Uはアルギニン(R)、グリシン(G)、セリン(S)、リジン(K)、トレオニン(T)、ロイシン(L)、アスパラギン(N)、ヒスチジン(H)、イソロイシン(I)から独立して選択されるアミノ酸残基である。
mは0、1、2または3であり、nは1または2であり、qは0、1、2または3である。 - 3~6アミノ酸残基からなる、請求項1に記載のペプチド。
- mが0または1であり、qが0または1である、請求項1または2に記載のペプチド。
- 配列番号1~18、配列番号65~71、NKP、KNP、NNP、INP、MNP、QNP、IKP、HKP、SKP、MKP、RKPのいずれかのアミノ酸配列を有する、請求項1~3のいずれか一項に記載のペプチド。
- 請求項1~4のいずれか一項に記載のペプチドと、有用タンパク質とを含む、タグ付加タンパク質。
- 有用タンパク質が酵素、サイトカイン、抗体、または蛍光タンパク質である、請求項5に記載のタグ付加タンパク質。
- 請求項5または6に記載のタグ付加タンパク質をコードするDNA。
- 請求項7に記載のDNAを含む組換えベクター。
- 請求項7に記載のDNAまたは請求項8に記載の組換えベクターで形質転換された形質転換体。
- 請求項9に記載の形質転換体を培養してタグ付加タンパク質を発現及び蓄積させ、タグ付加タンパク質を回収することを特徴とする、タグ付加タンパク質の製造方法。
- 請求項7に記載のDNAまたはそこから転写されるRNAを無細胞発現系に導入してタグ付加タンパク質を発現及び蓄積させ、タグ付加タンパク質を回収することを特徴とする、タグ付加タンパク質の製造方法。
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