WO2006059709A1 - 新規gstタグ配列及びその利用 - Google Patents
新規gstタグ配列及びその利用 Download PDFInfo
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- WO2006059709A1 WO2006059709A1 PCT/JP2005/022165 JP2005022165W WO2006059709A1 WO 2006059709 A1 WO2006059709 A1 WO 2006059709A1 JP 2005022165 W JP2005022165 W JP 2005022165W WO 2006059709 A1 WO2006059709 A1 WO 2006059709A1
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- protein
- amino acid
- recombinant protein
- acid sequence
- dartathione
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/23—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a GST-tag
Definitions
- the present invention relates to a wheat-derived dartathione-binding tag peptide, a recombinant protein obtained by fusing the tag peptide and a desired protein, a polynucleotide encoding the same, and a wheat germ extract, The use of the polynucleotide to express the recombinant protein.
- Glutathione-S-transferase (GST) fusion system is a system in which the target gene is incorporated in the 3'-side of the GST gene sequence on the vector in frame and expressed as a fusion protein in E. coli.
- the pGEX vector series is commercially available (Amersham Biosciences).
- the fusion protein is purified using affinity chromatography using a GST moiety (GST tag), and the target protein can be recovered after cleavage with a protease.
- a GST fusion protein is bound to a carrier (for example, dartathione sepharose 4B, etc.) to which DALTHATHION, which is a GST substrate, is bound, and the above-mentioned slag carrier is mixed with an elution buffer containing reduced DALTATHION.
- a carrier for example, dartathione sepharose 4B, etc.
- DALTHATHION which is a GST substrate
- Fusion protein expression using a GST tag can also be performed using a cell-free protein synthesis system, for example, wheat germ extract.
- a cell-free protein synthesis system for example, wheat germ extract.
- at least six GST families with different detoxifying activities against various herbicides have been reported in plant-derived GST families (for example, Non-patent Document 1). It is not clear whether or not these plants have a low homology with GST derived from Schistosoma japonicum, which is generally used as a GST tag, and bind to dartathione.
- Non-Patent Document 1 Dixon D. et al, The Journal of Biological Chemistry, 2003, Vol.278, No.26, pp.23930-23935
- an object of the present invention is to identify a dartathione-binding protein inherent in a wheat germ extract and provide an efficient gene expression and purification method for a fusion protein using the protein.
- the present inventors have found that there exists a protein (wGST) that exhibits the same behavior as a GST fusion protein when a protein expressed in a wheat germ extract is purified with a glutathione column.
- the amino acid sequence of this protein was determined. Based on the obtained amino acid sequence, the wGST is a protein having 222 amino acids from wheat belonging to the GST family, and a gene encoding the protein cloned from the wheat cDNA library As a result of expression in a cell-free system using wheat germ extract, it was found that wGST specifically binds to the dartathione column.
- the recombinant protein of the present invention has the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence.
- a tag peptide containing and having a dartathione binding activity is fused with a desired protein.
- the present invention provides a polynucleotide characterized by encoding the recombinant protein.
- the tag peptide in the recombinant protein is preferably encoded by DNA having the nucleotide sequence shown in SEQ ID NO: 1.
- the polynucleotide can be used to prepare a recombinant protein in a cell-free protein synthesis system using a wheat germ extract.
- the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence, and a dartathione bond
- the recombinant protein is preferably expressed in a cell-free protein synthesis system using wheat germ extract.
- novel GST tag sequence according to the present invention is a protein derived from wheat, it can be efficiently expressed in a cell-free system using a wheat germ extract, and can be used for purification of a fusion protein using a dartathione column. Is also suitable.
- FIG. 1 A comparison of the amino acid sequences of wGST, which is useful in the present invention, and GST, which has been used in the past.
- FIG. 2 shows the results of expression of wGST according to the present invention and GST-GFP used as a control experiment in a wheat germ extract and purification using dartathion oil.
- the dartathione-binding tag peptide of the present invention is a protein wG ST present in wheat germ, and was isolated using the binding activity to the dartathione column as an index, and the partial sequence of the amino acid was determined. As a result of searching the database using the obtained partial amino acid sequence, it was found that the protein was derived from wheat belonging to the GST family (GenBank Acc. No. CAC94002, SEQ ID NO: 2).
- FIG. 1 shows the homology between the wGST sequence and GST derived from Schistosoma japonicum that is generally used as a tag.
- the homology between these two sequences is clear whether wGST has dartathione-binding activity or not even with the sequence homology of as low as 20% in the 2Z3 region on the N-terminal side. is not.
- the 8 amino acid residues underlined on the C-terminal side of wGST in Fig. 1 are the partial sequences determined above. Therefore, the wGST cDNA was cloned from a wheat cDNA library, expressed in a wheat germ cell-free extract, and examined for its binding to GST. In a wheat germ cell-free expression system, The expression level is high, indicating that the expression level is equivalent to that of DHFR and that the purification efficiency is equal to or higher than that of conventional GST tags. won. Therefore, by combining a desired amino acid sequence to be expressed in wGST, a fusion protein that has high expression efficiency in a barley germ extract and can be easily purified using a dartathion column is provided.
- the recombinant protein of the present invention comprises the amino acid sequence shown in SEQ ID NO: 2 (wGST sequence), or an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence, and dartathione.
- a tag peptide having binding activity and a desired protein are fused.
- the number of deletions, substitutions or additions of the amino acid sequence is not particularly limited as long as the tag sequence has dartathione binding activity, but is usually 1 to 9, preferably 1 to 5, more preferably 1 to Two.
- the daltathione-binding tag peptide of the present invention has at least 50%, preferably 70%, 80%, 85%, 90%, 97%, 98% of the amino acid sequence shown in SEQ ID NO: 2 or the amino acid IJIJI. It may be a tag peptide having at least% homology and having dartathione binding activity.
- the degree of protein homology can be expressed as a percentage of identity when the amino acid sequences of the two proteins are properly aligned, and the appearance of an exact match between the sequences. Means rate. Appropriate alignment between sequences for identity comparison can be determined using various algorithms, such as the BLAST algorithm (Altschul SF J Mol Biol 1990 Oct 5; 215 (3): 403-10).
- the present invention also relates to a polynucleotide encoding the recombinant protein of the present invention.
- “Polynucleotide” generally refers to both polyribonucleotides or polydioxyribonucleotides, which may be modified. Examples thereof include DNA, cDNA, genomic DNA, mRNA, unprocessed RNA, and fragments thereof, and their length is not particularly limited.
- the “polynucleotide encoding the recombinant protein of the present invention” means that any nucleotide sequence based on the degeneracy of the genetic code can be selected as long as it can encode the recombinant protein of the present invention.
- DNAZRNA neurons may be double-stranded or single-stranded.
- DNAZRNA neurons are DNAZRNA neurons.
- These polynucleotides are chemically synthesized by various methods known to those skilled in the art. Alternatively, PCR can be performed by using all or part of cDNA or genomic DNA containing the polynucleotide as a cage. Thus, the polynucleotide can be amplified. It is also possible to synthesize mRNA by translating with SP6 RNA polymerase.
- a recombinant vector can be obtained by ligating (inserting) the polynucleotide of the present invention into an appropriate vector.
- the vector for inserting the polynucleotide of the present invention is not particularly limited as long as it can be replicated in the host, and examples thereof include plasmid DNA and phage DNA.
- Plasmid DNA includes plasmids derived from E. coli (eg, pRSET, pBR322, pBR325, pUC118, pUC119, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, etc.), and plasmids derived from yeast (eg, YEp13, YEp24).
- fage DNA include ⁇ phage (Charon4A, Charon21A, EMBL3, EMBL4, ⁇ gtlO, gtl1, ⁇ ZAP, etc.).
- animal winoles such as retrovirus or vaccinia virus and insect winoles vectors such as baculoinores can be used.
- the recombinant vector can express the recombinant protein of the present invention using the host by transforming the host cell.
- the host is not particularly limited as long as it can express the recombinant protein of the present invention. Examples thereof include bacteria such as coliforms, Bacillus subtilis, and Syudomonas. Also, Saccharomyces cerevisiae (3 ⁇ 4accharomyces cerevisiae pombe) and animal cells such as COS cells and CHO cells. Alternatively, insect cells such as Sf9 and Sf21 can be used.
- the recombinant protein of the present invention is preferably expressed using a cell-free protein synthesis system, particularly a wheat germ extract.
- a cell-free protein synthesis system particularly a wheat germ extract.
- a method for preparing such an extract is described in Madin K et al. Et al. (Madin K. et al., Proc. Natl. Acad. Sci. USA (2000), 97, 559-556,), Patent No. 3255784. , JP 2000-236896, WO00Z68412, Sawasaki T. et al. Et al. (Proc. Natl. Acad. Sci. USA (2002), 99, 14652-14657) and the like. [Method for purifying recombinant protein of the present invention]
- the recombinant protein of the present invention can be purified by affinity chromatography using, for example, a carrier (Dartathione 'Sepharose 4B) to which Dartathione, which is a substrate of GST, is bound via an epoxy bond. It can also be performed by a batch method, a column method, or a microspin method. Furthermore, the eluted fusion protein has a GST-tag sequence separated by a protease such as thrombin or active factor X by inserting a specific amino acid sequence between the tag peptide and the target protein in advance. It is also possible to isolate only the target protein. Reduced dartathione used for elution can be removed by a desalting column. Since the purification method of the present invention uses a GST tag peptide derived from wheat, it is particularly suitable for purification of a GST fusion recombinant protein synthesized in a cell-free system using a wheat germ extract.
- PCR was performed under the following conditions: 98 ° C for 1 minute (1 cycle), 98 ° C for 10 seconds, 58 ° C for 1 minute, 72 ° C for 3 minutes (30 cycles) ), 72 ° C for 5 minutes (1 cycle).
- the PCR product containing the amplified cDNA was incorporated into a pEU vector whose ends were treated with restriction enzymes using EcoRV and BamHI restriction enzyme sites. This is introduced into E. coli, amplified and The curry was extracted by the SDS method and prepared by density gradient centrifugation using a salt solution. Transcription was performed using SP6 polymerase with the prepared plasmid as a saddle type. The precipitate produced during the transcription reaction was removed by centrifugation, the transcription product in the supernatant was ethanol precipitated, air-dried, and used as a translation reaction type.
- the method for isolating intact (having germination ability) from seeds using mill, flotation, and sieve is the method of Johnston et al. (Johnston, FB et al. (1957) Nature, 179, 160-161). Improved and used. Chihoku wheat seeds (unsanitized) from Hokkaido were added to a mill (Fritsch Rotor Speed Mill pulverisette type 14) at a rate of 100 grams per minute, and the seeds were gently crushed at a rotation speed of 8000 rpm.
- the germinated embryos were collected from the floated fraction by flotation, and the organic solvent was removed by drying at room temperature. Impurities such as seed coats mixed in this germ fraction were removed, and wheat germ was selected visually.
- the selected wheat germ fraction is suspended in a 0.5% solution of Nodet P40 strength lye tester), and washed with an ultrasonic cleaner until the washing solution does not become cloudy. Wheat germ was purified.
- the following wheat germ extract was prepared according to a conventional method (Erickson, A. H. et al., (1996) Methods in Enzymology, Vol. 96, pp. 38-50). The following operations were performed at 4 ° C.
- a pure wheat straw embryo frozen in liquid nitrogen was ground in a mortar and 1 ml of the extracted solution obtained by partially modifying the method of Patterson et al. (80 mM HEPES-KOH, pH 7.6, 200 mM) Potassium acetate, 2 mM magnesium acetate, 4 mM calcium chloride, 8 mM dithiothreitol, 20 kinds each containing 0.6 mM L-type amino acid) were added, and the mixture was stirred with care not to generate bubbles.
- a translation reaction was performed by a dialysis method.
- Dialysis (Dialysis solution: final concentration 80OD, mRNA corresponding to transcription reaction solution 500, 40 ⁇ g / 1 creatine kinase, 30mM HEPES—KOH, pH 7.8, lOOmM potassium acetate, 2.7mM magnesium acetate, 4 mM dithiothreitol, 0.4 mM spermidine, 16 mM creatine phosphate, 0.3 mM 20 amino acids, 1.2 mM ATP, 0.25 mM ⁇ ) 500; ⁇ 1, dialysate (30 mM HEPES—KOH pH 7.8, lOOmM acetic acid rhodium, 2.7mM magnesium acetate, 4mM dithiothreitol, 0.4mM spermidine, 16mM creatine acid, 0.3mM 20 amino acids, 1.2mM ATP, 0.25mM GTP) , 26 °
- a fusion protein (GFP-GST) of GST derived from Schistosoma japonicum and green fluorescent protein (GFP) was used.
- GFP-GST fusion protein
- the dartathione resin used here was equilibrated in advance with a phosphate buffer. After sucrose was precipitated by centrifugation and the supernatant was removed, the phosphate buffer was added and stirred gently, and the supernatant was removed again by centrifugation. This process was repeated 4 times to remove unbound protein.
- Trit-HCl buffer (pH 8.0) / 10 mM reduced type dartathion was added to the dartathione resin precipitated in the centrifuge tube, gently stirred, centrifuged, and the supernatant was collected. After the protein recovered in the supernatant was separated by SDS-PAGE, specific binding to dartathione resin was confirmed by CBB staining.
- lane 1 is the molecular weight marker
- lanes 2 to 4 are the GST-GFP used as a control experiment
- the protein in the translation reaction (lane 2), and the unbound protein recovered from the supernatant of the dartathione resin.
- (Lane 3) and the protein eluted by adding reduced dartathione (Lane 4).
- lanes 5-7 the wGST synthesized above is supplemented with the protein in the translation reaction solution (lane 5), the unbound protein recovered from the supernatant of dartathion resin (lane 6), and reduced dartathione. Shown is the protein (lane 7) that was eluted in a column.
- wGST has the same or higher ability to bind to fat as GST GFP and is eluted from the fat by reduced glutathione like GST-GFP. Therefore, wGST is one In general, the GST and the purification process show the same behavior, so the possibility of being used as a tag has increased.
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Cited By (1)
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CN102524514A (zh) * | 2012-01-18 | 2012-07-04 | 安徽燕之坊食品有限公司 | 一种小麦胚芽活性多肽的制备方法 |
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WO1999014337A2 (en) * | 1997-09-16 | 1999-03-25 | Rhone-Poulenc Agriculture Limited | New plant genes |
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WO1999014337A2 (en) * | 1997-09-16 | 1999-03-25 | Rhone-Poulenc Agriculture Limited | New plant genes |
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CN102524514A (zh) * | 2012-01-18 | 2012-07-04 | 安徽燕之坊食品有限公司 | 一种小麦胚芽活性多肽的制备方法 |
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