JP2002371096A - Method for selectively cutting fusion protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a selective cutting method for obtaining a desired protein from a fusion protein, and a method for obtaining the desired protein from the fusion protein by enzymatically cutting an amino acid sequence in a fusion part or its peripheral part. SOLUTION: A process in which, from the fusion protein prepared by linking a tug and a protein, the desired protein is obtained by cutting the linking part or its peripheral part without cutting the sequence of the protein part. In the process, a nonspecific endprotease is used.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for cleaving a fusion protein in which a tag and a protein are linked with a non-specific endoprotease. Further, the present invention relates to a selective cleavage method for obtaining a desired protein from a fusion protein. Further, the present invention relates to a method for obtaining a desired protein from a fusion protein, which comprises enzymatically cleaving an amino acid sequence of a connecting portion and a peripheral portion thereof. The present invention
It is useful for fusion proteins and the like that are difficult to selectively cut by ordinary methods. Further, according to the present invention, it becomes possible to prepare a desired protein from a fusion protein at a very low cost and easily.

[0002]

2. Description of the Related Art Advances in genetic engineering have made it possible to analyze proteins that have been purified from natural products at the gene level and artificially amplify the target protein. Thioredoxin invented thereafter (Tokuhyohei 5-507209)
And glutathione-S-transferase (GST)
By applying a DNA sequence obtained by fusing (for example, Japanese Patent Application Laid-Open No. 1-503441), a protein that is originally difficult to express can be expressed, and techniques for expressing a fusion protein have been widely used. Was. Furthermore, these fusion proteins can be easily purified by using a compound that binds to the fused peptide as a carrier, and the purification step can be greatly simplified. For such a purpose, various peptides (tags) having a specific amino acid sequence for use in preparing a fusion protein have been developed. As a result, an affinity peptide containing an adjacent histidine residue (His-tag) (JP-A-63-2
51095) and maltose binding protein (JP-A-1-20
094) and a desired protein through a linking portion, and a technique for easily purifying the prepared fusion protein using affinity chromatography or the like has been established. However, in order to obtain a desired protein from the fusion protein, it is necessary to cleave and remove the fused peptide. When the fusion protein has substantially the same properties and biological activity as the desired protein, the fusion protein can be used for application as it is, but such a case is rare. Many fusion proteins are affected by the fused peptide, and its properties and bioactivity are notably changed. As a method for removing the fused peptide, a method of enzymatic cleavage and a method by chemical reaction are considered, but in order to obtain a desired protein in a stable state, a method of enzymatic cleavage is generally adopted. You. The enzymatic cleavage method is a method in which a recognition amino acid sequence of a sequence-specific protease is previously inserted into a linking portion between a desired protein and a fused peptide, and the corresponding sequence-specific protease is allowed to act on the fused peptide. It is a method of removing. A sequence-specific protease is an enzyme capable of recognizing and cleaving only a specific amino acid sequence, and has no fear of cleaving a desired protein. However, sequence-specific proteases are very expensive, and it is not practical to use this method to create practical enzymes, and a new method that is inexpensive and has the same effect and simplicity as sequence-specific proteases is expected. Was rare.

[0003]

For these reasons,
There has been a demand for a method for inexpensively and conveniently performing selective cleavage of a fusion protein. That is, an object of the present invention is to provide a method for selectively cleaving a fusion protein by an inexpensive and simple method.

[0004]

Means for Solving the Problems We have found that it is possible to cleave a fusion protein in which a tag and a protein are linked by using a non-specific endoprotease, and have reached the present invention. More specifically, they have found that by using a non-specific endoprotease, it is possible to obtain a useful protein from a fusion protein having a specific amino acid sequence in the amino acid sequence of the connecting portion, and thus have accomplished the present invention. Heretofore, non-specific endoproteases have been considered not to recognize a specific sequence and to cleave the protein at a plurality of sites, so that it has been considered impossible to use the method for selectively cleaving the fusion protein. However, we have conducted various studies, and have made it possible to obtain a useful protein from a fusion protein having a specific amino acid sequence at the junction using a non-specific endoprotease.

That is, the present invention has the following configuration. [1] A method in which a fusion protein in which a tag and a protein are linked is cleaved with a non-specific endoprotease. [2] In a step of cleaving the sequence of the linking portion or its peripheral portion without cleaving the sequence of the protein portion to obtain the desired protein, the non-specific endoprotease is used for the fusion protein obtained by linking the tag and the protein. A method for obtaining a useful protein from a fusion protein, characterized by using: [3] The method according to [2], wherein the amino acid sequence of the connecting portion is represented by the formula (1). X-Met-Phe-Ser- (Xaa) nY (1) wherein X represents a protein and Y represents a tag. Xaa means any amino acid residue. n is an integer of 0 to 50. [4] The method according to [2], wherein the amino acid sequence of the connecting portion is represented by the formula (2). X-Met-Phe-Ser-Asp-Glu-Pro-Asp-His-Lys-Gly-Ala-Leu-
Lys- (Xaa) nY (2) [wherein, X represents a protein and Y represents a tag. Xaa means any amino acid residue. n is an integer of 0 to 40. [5] The method according to [2], wherein the amino acid sequence of the connecting portion is represented by the formula (3). X-Met-Phe-Ser-Asp-Glu-Pro-Asp-His-Lys-Gly-Ala-Leu-
Lys-Y (3) [where X represents a protein and Y represents a tag. [6] The method according to [1] to [5], wherein the tag is an affinity peptide containing an adjacent histidine residue. [7] The protein is an enzyme [1]-
The method according to [6]. [8] the enzyme is uricase [7]
The method described in. [9] The method of [8], wherein the uricase is derived from Bacillus sp. TB90. [10] The method according to [1] to [9], wherein the non-specific endoprotease is selected from proteinase K, trypsin, chymotrypsin, and V8 protease. [11] The method according to [10], wherein the non-specific endoprotease is proteinase K.

[0006] In the present invention, the non-specific endoprotease refers to a protease capable of cleaving a protein at a plurality of positions without recognizing a specific sequence. Specifically, when reacted with a protein, it does not recognize a specific sequence and cleaves the protein at a plurality of sites, but when reacted with a fusion protein of the protein according to the present invention, without cutting the sequence of the protein portion, It refers to a protease that cleaves the sequence of the connecting portion or its peripheral portion.

More specifically, it is a protease having any of the following properties. For example, (a) a yield of 60% or more when 1 g / L protease is reacted with 3 g / L protein in 20 mM, pH 7.5 Tris-HCl buffer at 37 ° C. for 30 minutes, (B) a protease having such a property that the yield of the desired protein exceeds 40% when reacted with the fusion protein of the protein according to the present invention under the same conditions. Preferably, in (a), the protease has a property such that the yield of the protein is 80% or more, but in (b), the yield of the desired protein according to the present invention exceeds 70%, and more preferably. , (A) is a protease having a protein yield of 95% or more, while (b) is a protease having such a property that the desired protein yield of the present invention exceeds 90%. The yield can be determined by measuring the activity of an enzyme or the like, or by an immunoassay. That is, the yield can be determined by determining the ratio of the activity or the immunoassay value before and after the treatment with the protease.

[0008] Alternatively, for example, 1 g / L of protease is added to 3 g / L in 20 mM, pH 7.5 Tris-HCl buffer.
This protease has a property such that when reacted with the L protein at 37 ° C. for 30 minutes, the ratio of the largest amount of the decomposed products to the total of the decomposed products does not exceed 40%. Preferably, the protease has such a property that the ratio of the most abundant degradation products to the whole does not exceed 20%. Even more preferred is a protease having such a property that the ratio of the most abundant degradation products to the whole does not exceed 10%. The ratio of the decomposition products is, for example, SDS-PAG.
It can be obtained by calculation from data such as E. That is, the ratio can be determined by applying the result of SDS-PAGE of the reaction solution to a one-dimensional image analyzer and a densitometer and calculating the concentration of each decomposition product band as a numerical value.

The nature of the protein of interest in the method of the present invention is not particularly limited, but is preferably a single protein having a specific molecular weight.

[0010] The method for selectively cleaving a fusion protein in the present invention is preferably carried out by the following steps. That is, (1) preparation of a gene encoding a fusion protein in which a tag and a target protein are linked. (2) Synthesis of fusion protein. (3) Preparation of fusion protein. (4) Enzymatic degradation of the sequence at the junction of the fusion protein or its surroundings. (5) Preparation of desired protein. In addition, even if the steps (4) and (5) are summarized, substantially the same result may be obtained in some cases.

In the present invention, the means for synthesizing the fusion protein used is not particularly limited. As the method, a conventional genetic engineering technique, a modification thereof, a protein engineering technique, and the like can be used. In particular,
The tag and the protein can be ligated at the gene level, and the produced fusion protein gene can be synthesized by expressing it in a recombinant bacterium using a gene recombination technique. Furthermore, it is possible to increase the purity of the fusion protein by using culture and purification techniques, and to obtain a substantially single protein sample. A general method for obtaining a target protein using a genetic manipulation system of a microorganism is described below.

The method for obtaining the gene of the protein of the present invention includes, for example, a method in which chromosomal DNA is separated and purified, and then the DNA is cut by sonication, treatment with a restriction enzyme, or the like;
The recombinant expression vector is constructed by binding and closing the linear expression vector at the blunt end or cohesive end of both DNAs with DNA ligase or the like. After the thus obtained recombinant vector is transferred to a replicable host microorganism, screening is performed using the expression of the marker and the enzyme activity of the vector as an indicator, and the microorganism carrying the recombinant vector containing the gene encoding the desired protein is screened. Get. Next, the microorganism is cultured, the recombinant vector is separated and purified from the cultured cells, and the gene of the desired protein may be collected from the recombinant vector.

[0013] The chromosomal DNA is specifically collected as follows. That is, for example, a culture obtained by stirring and culturing the donor organism for 1 to 3 days is collected by centrifugation, and then lysed to prepare a lysate containing the gene. As a method of lysis, for example, treatment with a lytic enzyme such as lysozyme or β-glucanase is performed, and if necessary, a protease or another enzyme or a surfactant such as sodium lauryl sulfate (SDS) is used in combination. Or a physical crushing method such as a French press treatment. DNA is separated and purified from the lysate obtained in this manner by appropriately combining methods such as deproteinization by phenol treatment or protease treatment, ribonuclease treatment, or alcohol precipitation, according to a conventional method. Can be. The method of cutting the separated and purified DNA can be performed by, for example, ultrasonic treatment, restriction enzyme treatment, or the like. Preferably, a type II restriction enzyme that acts on a specific nucleotide sequence is suitable.

As the vector, a vector constructed for gene recombination from a phage or a plasmid capable of autonomous growth in a host microorganism is suitable. As the phage, for example, Escherichia coli
When i) is used as a host microorganism, Lambda-gt10, Lambda
-gt11 etc. can be used. As a plasmid, for example, when Escherichia coli is used as a host microorganism, pBR322, pUC19, pBluescript, pLE
DM1 (Journal of Fermentation and Bioenzineerin
g, Vol. 76, 265-269 (1993)). Such a vector can be cut with the restriction enzyme used for cutting the above-mentioned stained DNA to obtain a vector fragment, but it is not always necessary to use the same restriction enzyme as the restriction enzyme used for cutting the chromosomal DNA. . A method for joining a chromosomal DNA fragment and a vector DNA fragment is known in the art.
Any method using NA ligase may be used. For example, after annealing the cohesive end of the chromosomal DNA fragment and the cohesive end of the vector fragment, a recombinant vector of the chromosomal DNA fragment and the vector DNA fragment is prepared by using an appropriate DNA ligase. I do. If necessary, after annealing, it can be transferred to a host microorganism and a recombinant vector can be prepared using in vivo DNA ligase.

As the host microorganism, any microorganism can be used as long as the recombinant vector is stable, capable of autonomous propagation and capable of expressing a foreign gene, and generally Escherichia coli W3
110, Escherichia coli C600, Escherichia coli HB101, Escherichia coli JM109 and the like can be used. As a method for transferring the recombinant vector to the host microorganism, for example, when the host microorganism is Escherichia coli, a competent cell method or an electroporation method by calcium treatment can be used.
The microorganism, which is a transformant thus obtained, can stably produce a large amount of protein by being cultured in a nutrient medium. The selection as to whether or not the target recombinant vector has been transferred to the host microorganism may be made by searching for a microorganism that expresses a drug resistance marker of the vector holding the target DNA. The recombinant vector obtained by the above method can be easily removed from a transformed microorganism and transferred to another microorganism. In addition, DN, which is a gene encoding a protein by restriction enzyme or PCR,
A can be easily collected, ligated to another vector fragment, and transferred to a host microorganism.

The culture form of the host microorganism, which is a transformant, may be selected by taking into consideration the nutritional and physiological properties of the host. Usually, liquid culture is used in many cases. Advantageously, a culture is performed. As the nutrient source of the medium, those commonly used for culturing microorganisms can be widely used. The carbon source may be any carbon compound that can be assimilated, such as glucose, sucrose, lactose, maltose, fructose, molasses, and pyruvic acid. Any nitrogen compound can be used as the nitrogen source, and examples thereof include peptone, meat extract, yeast extract, casein hydrolyzate, and soybean meal alkaline extract. In addition, salts such as phosphate, carbonate, sulfate, magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, specific vitamins, and the like are used as necessary. The culture temperature can be appropriately changed within a range in which the bacteria grow and produce the target protein. In the case of Escherichia coli, the temperature is preferably about 20 to 42 ° C. The cultivation time varies somewhat depending on the conditions, but the cultivation may be terminated at an appropriate time in consideration of the time at which the target protein reaches the maximum yield, and is usually about 6 to 48 hours. Medium pH
The pH can be appropriately changed within a range in which the bacterium grows and produces the target protein, and the pH is particularly preferably about 6.0 to 9.0.

The culture solution containing the cells producing the desired protein in the culture can be directly collected and used.
In general, when a target protein is present in a culture solution according to a conventional method, it is used after separating a solution containing the target protein from microbial cells by filtration, centrifugation, or the like. If the desired protein is present in the cells, collect the cells from the resulting culture by filtration or centrifugation, and then destroy the cells by a mechanical method or an enzymatic method such as lysozyme. And, if necessary, EDT
A target protein is solubilized by adding a chelating agent such as A and / or a surfactant, and separated and collected as an aqueous solution.

The solution containing the target protein thus obtained is concentrated, for example, under reduced pressure, membrane concentration, salting-out treatment with ammonium sulfate, sodium sulfate or the like, or a hydrophilic organic solvent such as methanol, ethanol, acetone or the like. May be precipitated by the fractional precipitation method described above. Heat treatment and isoelectric point treatment are also effective purification means. Thereafter, gel filtration using an adsorbent or a gel filtration agent, adsorption chromatography, ion exchange chromatography, and affinity chromatography are performed to obtain a purified target protein. For example, Sephadex G-25 (Pharmacia
Gel filtration by biotech), DEAE Sepharose CL
-6B (Pharmacia Biotech) and Octyl Sepharose CL6-B (Pharmacia Biotech) can be separated and purified by column chromatography to obtain a purified enzyme preparation. This purified enzyme preparation has been purified to the extent that it shows almost a single band by electrophoresis (SDS-PAGE).

The fusion protein may be synthesized using a cell-free protein synthesis method. BACKGROUND ART With the progress of biotechnology in recent years, protein synthesis using a cell-free protein synthesis method has been rapidly attracting attention. Cell-free protein synthesis removes components related to protein synthesis in the living body out of the living body, and extracts mRNA encoding the target protein.
It is a means to synthesize proteins semi-artificially by adding energy sources such as ATP and GTP. As the biomaterial, Escherichia coli, reticulocytes, and wheat germ are often used, and various preparation methods and utilization methods have been studied so far. The advantages of cell-free protein synthesis over protein synthesis using recombinant microorganisms or cultured cells are:
1) It is relatively easy to synthesize proteins that have a negative effect on living organisms (excluding those that affect the translation system). 2) Conditions can be determined easily. It usually takes about one month), 3)
Unnatural amino acids can be used.

In the present invention, the origin of the sequence non-specific protease used for enzymatically decomposing the sequence of the fusion protein at the junction or its periphery is not particularly limited, but it belongs to serine protease, thiol protease and the like. It is preferable to use proteases, specifically, proteinase K, trypsin, chymotrypsin, subtilisin, V8 protease and the like. The conditions of the decomposition reaction by the protease depend on the enzymatic properties of each protease, but generally, the pH of the reaction solution is fixed with a buffer such as a GOOD buffer or a phosphate buffer, and the pH is 10 to 90.
Incubate at 5 ° C for 5 minutes to 24 hours. For details, for example,
Neutral protease to weakly acidic to weakly alkaline Tris-HCl
Incubate in buffer at 20-80 ° C for 10 minutes to 16 hours. Alternatively, alkaline protease is added at pH 8-11.
30 minutes to 16 minutes at 10 to 60 ° C. in GOOD buffer
Let react for hours.

The solution of the fusion protein used in the present invention may be used as it is after synthesis, but may be purified. Preferably, purification is performed to high purity using ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration, hydroxyapatite, or isoelectric focusing. More preferably, thioredoxin, GST, Hi
Affinity purification can also be performed by a purification method utilizing specific recognition and adsorption of a peptide having a specific amino acid sequence to be fused, such as s-tag and maltose binding protein.

The protein used in the present invention is not limited to an enzyme, and may be any protein whose existence can be confirmed in some form.
Enzymes include uricase, catalase, amine oxidase, glucose oxidase, amino acid dehydrogenase, DNA polymerase, DNA ligase, restriction enzymes,
DNA as a protein whose existence can be confirmed, such as protein kinase, protease, lipase, amylase, etc.
Examples include binding proteins, receptor proteins, chaperonins, cytokines, hormone-like proteins, and the like.

One embodiment of the present invention is a method for easily and inexpensively obtaining a desired protein from a fusion protein. Another embodiment of the present invention is a reagent system for studying protein purification and structural analysis. By using the present invention, it may be possible to easily prepare a protein, which has been difficult to purify at low cost with conventional techniques. In addition, it becomes possible to efficiently prepare a target protein in supplying materials for structural analysis.

[0024]

EXAMPLES The effects of the present invention will be further clarified by exemplifying embodiments of the present invention, but the present invention is not limited thereto. Example 1 Preparation of Gene Encoding Fusion Protein As a desired protein, uricase (uric acid oxidase, EC 1.7.3.3) derived from Bacillus sp. ). As a peptide having a specific amino acid sequence, an affinity peptide containing an adjacent histidine residue, specifically, 6xHis-tag in which six histidine residues are arranged, is bound to the carboxyl terminus of uricase via a linking moiety. The gene encoding the fusion protein thus prepared was prepared according to the following procedure. The creation flow is also shown in FIG. Plasmid pUOD containing uricase gene (Japanese Unexamined Patent Publication No.
3488 PR) (Journal of Biochemistry, Vol. 119,
80-84 (1996)) (Bioindustry, Vol.13, 20-
28 (1996)) as a template, a uricase gene for preparing a fusion protein gene was prepared by performing polymerase chain reaction (PCR) under the following composition and conditions. DNA polymerase (KOD-Plus- (manufactured by Toyobo)) 1 μl Template (pUOD) 0.05 ng Primer (1) (described in SEQ ID NO: 1 in the Sequence Listing) 25 pmole Primer (2) (described in SEQ ID NO: 2 in the Sequence Listing) 25 pmole × 10 buffer (Manufactured by Toyobo) 5 μl 2 mM dNTP (manufactured by Toyobo) 5 μl 25 mM MgSO4 (manufactured by Toyobo) 4 μl Total liquid volume: 50 μl PCR conditions: 94 ° C., 2 minutes ↓ 94 ° C., 15 seconds → 50 ° C., 30 seconds → 68 ° C., 1 minute (30 cycles) ↓ 68 ° C, 2 minutes The sequence of the uricase gene obtained as a PCR product is described in SEQ ID NO: 3 in the Sequence Listing, and the amino acid sequence of uricase is described in SEQ ID NO: 4 in the Sequence Listing. The obtained uricase gene is used as a PCR product purification kit (MagExtractor-PCR
& Gel Clean Up- (Toyobo)), purify according to the protocol, cut with restriction enzymes EcoRI and PstI, and similarly
And the expression vector pKK223-3 (Amersham Pharmacia) cut with PstI and ligated using T4 DNA ligase. The resulting uricase gene expression plasmid was named pUODSP1 (FIG. 1). pUO
DSP1 was digested with restriction enzymes SmaI and PstI to cleave the carboxyl terminal side of the uricase gene. Synthetic oligonucleotides having the sequences shown in SEQ ID NOs: 5 and 6 in the sequence listing were ligated to this using T4 DNA ligase, and finally 6xHis-tag in which six histidine residues were arranged was linked to the carboxyl terminus of uricase. Thus, an expression plasmid containing the gene encoding the fusion protein linked via the DNA, pUODSP1-HT, could be prepared (FIG. 1). The sequence of the gene encoding the fusion protein thus prepared is described in SEQ ID NO: 7 of the Sequence Listing, and the amino acid sequence of the fusion protein is described in SEQ ID NO: 8 of the Sequence Listing.

Example 2 Preparation of fusion protein Competent cells of Escherichia coli JM109 (Competent High (manufactured by Toyobo)) were transformed with pUODSP1-HT, and a fusion protein-producing recombinant bacterium JM109 / pUODSP was prepared.
1-HT was prepared. 50 ml TB-brot
h. Inoculate 500 ml volumetric flask containing
The cells were cultured for 7 hours. The cells are collected from the culture solution by centrifugation, and buffer A (20 mM Tris-HCl (pH 7.
5) After suspending in 350 mM NaCl), proteins were extracted by sonication. Nickel chelate
Through column chromatography (HiTrap-Chelating- (Amersham Pharmacia)), buffer B (20 mM Tris-H) was used according to the manufacturer's protocol.
Cl (pH 7.5), 350 mM NaCl, 500 m
M imidazole). Further, the eluate is
The solution was passed through phadex G-25 desalting / column chromatography (Hitrap-Desalting- (Amersham Pharmacia)), and eluted with buffer C (20 mM Tris-HCl (pH 7.5)) according to the manufacturer's protocol. A purified preparation of the fusion protein was prepared. FIG. 2 shows the results of analysis of the disrupted supernatant and the purified protein by SDS-polyacrylamide gel.

Example 3 Treatment with Non-Specific Protease and Preparation of Desired Protein Purified fusion protein was purified from 20 mM Tris-HCl (pH 7.0) of Proteinase K (manufactured by Nacalai Tesque, Inc.).
5) Treated with solution. 50 μl fusion protein: about 2 mg
/ Μl to proteinase K: about 0.1 to 1 μg / m
and a treatment at 37 ° C. for 30 minutes. The reaction was terminated by adding PMSF (phenylmethylsulfonyl fluoride (manufactured by Nacalai Tesque)) as a proteinase K inhibitor for a final concentration of 1 mM. As a result, the carboxyl terminus of the fusion protein was selectively cleaved with the treatment concentration of proteinase K, and uricase, a desired protein, could be obtained. FIG. 2 shows the results of analysis of the fusion protein-treated proteinase K product by SDS-polyacrylamide gel. The junction of the fusion protein was cleaved by the enzymatic degradation of proteinase K, resulting in 6 × H
The analysis result revealed that the is-tag peptide was removed and uricase was being prepared. Cleavage of the junction of the fusion protein by a non-specific protease
It was also confirmed by mass spectrometry using LDI-TOFF-MASS. It is presumed that the selective cleavage of the fusion protein by the non-specific protease is due to the structural features of the junction or the junction and its surroundings, but at this time, the detailed basic mechanism has not been clarified. Only its usefulness in terms has been found. Therefore, X-Met-Phe-Ser- (Xaa) nY (1) wherein the amino acid sequence of the connecting portion is represented by the formula (1) [where X represents a protein having a specific molecular weight. Y means a peptide having a specific amino acid sequence. Xaa means any amino acid residue. n is an integer of 0 to 50. Or X-Met-Phe-Ser-Asp-Glu-Pro-Asp-His-Lys-Gly-Ala-Leu-, wherein the amino acid sequence of the connecting portion is represented by the formula (2).
Lys- (Xaa) nY (2) wherein X represents a protein having a specific molecular weight, and Y represents a peptide having a specific amino acid sequence. Xaa means any amino acid residue. n is an integer of 0 to 40. Or X-Met-Phe-Ser-Asp-Glu-Pro-Asp-His-Lys-Gly-Ala-Leu-, wherein the amino acid sequence of the connecting portion is represented by the formula (3).
Lys-Y (3) wherein X represents a protein having a specific molecular weight, and Y represents a peptide having a specific amino acid sequence. ] Information as a novel method has been disclosed as an example.

Example 4 Affinity of fusion protein
Preparation of desired protein by binding to beads and non-specific protease treatment By using the method of this patent, the fusion protein, which has been practically difficult in terms of cost, can be bound to affinity beads and sequence-selectively cleaved. Thus, a method of obtaining a desired protein becomes possible. By enabling this technique, high-throughput purification of proteins can be easily performed. FIG. 3 shows an example of a flow of high-throughput purification of a protein according to the method of the present patent. As in Example 2,
The fusion protein producing recombinant bacterium JM109 / pUODSP1-HT was inoculated into TB-broth and cultured at 37 ° C. for 17 hours. The cells are collected from the culture solution by centrifugation and buffer A
After suspension in, the protein was extracted by sonication. Dissolve the crushed liquid with nickel chelate magnetic beads (MagExtractor-His
-tag- (manufactured by Toyobo), and the beads were washed with an attached buffer according to the manufacturer's protocol. By this operation, magnetic beads to which the fusion protein has been adsorbed are prepared. The magnetic beads were mixed with 20 mM Tris-HCl (pH 7.0) of Proteinase K (manufactured by Nacalai Tesque, Inc.).
5) The solution was treated to elute the desired protein. 100 μl of proteinase K solution was added to a volume of 20 μl of the fusion protein-adsorbed magnetic beads at a concentration of about 1 to 4 μg / ml, followed by treatment at 37 ° C. for 60 minutes. As a result, the carboxyl terminus of the fusion protein was selectively cleaved with the treatment concentration of proteinase K, and uricase, a desired protein, could be obtained. Proteinase K of magnetic beads
FIG. 3 shows the results of analysis of the treated product by SDS-polyacrylamide gel. The junction of the fusion protein was cleaved by the enzymatic degradation of proteinase K, resulting in 6 × Hi
The analysis results revealed that the desired protein was eluted with the s-tag peptide adsorbed on the magnetic beads, and a solution containing the desired protein was prepared by solid-liquid separation. Cleavage of the junction of the fusion protein by non-specific protease was also confirmed by mass spectrometry using MALDI-TOFF-MASS. In this embodiment, since magnetic beads are used, solid-liquid separation can be performed very easily using a magnet without performing centrifugation. The solid-liquid separation system for magnetic beads using magnets is easy to automate, and in fact, automation equipment has already been developed,
Sold (MFX-2000, MFX-6000, MFX-9600 (manufactured by Toyobo)). Therefore, by combining these devices with the method of the present patent, high-throughput purification of proteins can be easily realized.

[0028]

According to the present invention, selective cleavage of a fusion protein by a non-specific protease, which has been difficult with conventional techniques, becomes possible. This makes it possible to easily and inexpensively prepare a desired protein from the fusion protein.
In addition, taking advantage of the cost advantage of the present method, it can be expected to be applied to high-throughput screening of proteins, which was conventionally difficult in terms of cost.

[0029]

[Sequence List] <110> Toyo Boseki Kabushiki Kaisya <120> Specific cleavage method of fusion protein <130> 01-0459 <141> 2001-06-18 <160> 8 <170> PatentIn version 2.0 <210> 1 <211 > 21 <212> DNA <213> Artificial sequence <220> <223> the sequence of designed polynucleotide for PCR primer, describedin example no.1 <400> 1 aaacagaatt cgagctcgcg c 21

<210> 2 <211> 41 <212> DNA <213> Artificial sequence <220> <223> the sequence of designed polynucleotide for PCR primer, describedin example no.1 <400> 2 gatatctgca gtcacccggg tttaagtgct cctttatgat c 41

<210> 3 <211> 1083 <212> DNA <213> Bacillus sp.TB-90 <400> 3 aaacagaatt cgagctcgcg caatgtttcg tttgcaagaa atatttcgtg aaggagagaa 60 taattcg atg acc aaa cac aaa gaa aga gt g atg tat gat tat 109 Met Thr Lys His Lys Glu Arg Val Met Tyr Tyr Gly Lys Gly 1 5 10 gac gta ttt gct tat cgc acc tat tta aaa cca ctt act gga gtt aga 157 Asp Val Phe Ala Tyr Arg Thr Tyr Leu Lys Pro Leu Thr Gly Val Arg 15 20 25 30 acg att cct gaa tct cca ttt tcc ggt cga gat cat att ctt ttt gga 205 Thr Ile Pro Glu Ser Pro Phe Ser Gly Arg Asp His Ile Leu Phe Gly 35 40 45 gta aat gta aaa atc tca gta gga gga aca aaa ttg ctg acc tcc ttt 253 Val Asn Val Lys Ile Ser Val Gly Gly Thr Lys Leu Leu Thr Ser Phe 50 55 60 acg aaa ggg gat aac agc tta gtc gtt gca aca gac tcg atg aaa aac 301 Thr Lys Gly Asp Asn Ser Leu Val Val Ala Thr Asp Ser Met Lys Asn 65 70 75 ttt ata caa aaa cat tta gct agt tat aca gga aca acg ata gaa ggt 349 Phe Ile Gln Lys His Leu Ala Ser Tyr Thr Gly Thr Thr Ile Glu Gly 80 85 90 ttt tta gaa tat gta gct act tct ttt ttg aag aaa tat tct cat att 397 Phe Leu Glu Tyr Val Ala Thr Ser Phe Leu Lys Lys Tyr Ser His Ile 95 100 105 110 gaa aag att tcg ttg ata gga gag gaa att ccc ttt gaa aca act ttt 445 Glu Lys Ile Ser Leu Ile Gly Glu Glu Ile Pro Phe Glu Thr Thr Phe 115 120 125 gca gta aag aat gga aat aga gca gct agt gag cta gta ttt aaa aaa 493 Ala Val Lys Asn Gly Asn Arg Ala Ala Ser Glu Leu Val Phe Lys Lys 130 135 140 tca cga aat gaa tat gcc acc gct tat ttg aat atg gtt cgt aat gaa 541 Ser Arg Asn Glu Tyr Ala Thr Ala Tyr Leu Asn Met Val Arg Asn Glu 145 150 155 gat aac acc cta aac att act gaa caa caa agc gga ctt gct ggt ctt 589 Asp Asn Thr Leu Asn Ile Thr Glu Gln Gln Ser Gly Leu Ala Gly Leu 160 165 170 caa tta ata aaa gtc agc gga aat tcc ttt gtc ggt ttt att cgt gac 637 Gln Leu Ile Vals Ser Gly Asn Ser Phe Val Gly Phe Ile Arg Asp 175 180 185 190 gaa tac aca act ctt cca gag gat tca aac cgc cct cta ttt gtt tac 685 Glu Tyr Thr Thr Leu Pro Glu Asp Ser Asn Arg Pro Leu Phe Val Tyr 195 200 Two 05 tta aac atc aaa tgg aag tac aaa aac acg gaa gac tca ttt gga acg 733 Leu Asn Ile Lys Trp Lys Tyr Lys Asn Thr Glu Asp Ser Phe Gly Thr 210 215 220 aat cca gaa aat tat gtt gca gct gaa caa att cgcac atc gcc act 781 Asn Pro Glu Asn Tyr Val Ala Ala Glu Gln Ile Arg Asp Ile Ala Thr 225 230 235 tcc gta ttt cat gaa acc gag acg ctt tcc atc caa cat tta att tat 829 Ser Val Phe His Glu Thr Glu Thr Leu Ser Ile Gln His Leu Ile Tyr 240 245 250 tta atc ggc cgc aga ata tta gaa aga ttc cct caa ctt caa gaa gtt 877 Leu Ile Gly Arg Arg Ile Leu Glu Arg Phe Pro Gln Leu Gln Glu Val 255 260 265 270 270 tac ttcga caa aat cat aca tgg gat aaa ata gtg gag gaa att 925 Tyr Phe Glu Ser Gln Asn His Thr Trp Asp Lys Ile Val Glu Glu Ile 275 280 285 cct gaa tca gaa ggg aaa gta tat aca gaa ccg cga ccg cca tat g Glu Ser Glu Gly Lys Val Tyr Thr Glu Pro Arg Pro Pro Tyr Gly 290 295 300 ttt caa tgc ttt act gtc acc caa gaa gac ttg cca cac gaa aac att 1021 Phe Gln Cys Phe Thr Val Thr Gln Glu Asp Leu Pro His Glu AsnIle 305 310 315 ctt atg ttc tct gat gaa ccc gat cat aaa gga gca ctt aaa ccc ggg 1069 Leu Met Phe Ser Asp Glu Pro Asp His Lys Gly Ala Leu Lys Pro Gly 320 325 330 tgactgcaga tatc 1083

<210> 4 <211> 334 <212> PRT <213> Bacillus sp.TB-90 <400> 4 Met Thr Lys His Lys Glu Arg Val Met Tyr Tyr Gly Lys Gly Asp Val 1 5 10 15 Phe Ala Tyr Arg Thr Tyr Leu Lys Pro Leu Thr Gly Val Arg Thr Ile 20 25 30 Pro Glu Ser Pro Phe Ser Gly Arg Asp His Ile Leu Phe Gly Val Asn 35 40 45 Val Lys Ile Ser Val Gly Gly Thr Lys Leu Leu Thr Ser Phe Thr Lys 50 55 60 Gly Asp Asn Ser Leu Val Val Ala Thr Asp Ser Met Lys Asn Phe Ile 65 70 75 80 Gln Lys His Leu Ala Ser Tyr Thr Gly Thr Thr Ile Glu Gly Phe Leu 85 90 95 Glu Tyr Val Ala Thr Ser Phe Leu Lys Lys Tyr Ser His Ile Glu Lys 100 105 110 Ile Ser Leu Ile Gly Glu Glu Ile Pro Phe Glu Thr Thr Phe Ala Val 115 120 125 Lys Asn Gly Asn Arg Ala Ala Ser Glu Leu Val Phe Lys Lys Ser Arg 130 135 140 Asn Glu Tyr Ala Thr Ala Tyr Leu Asn Met Val Arg Asn Glu Asp Asn 145 150 155 160 Thr Leu Asn Ile Thr Glu Gln Gln Ser Gly Leu Ala Gly Leu Gln Leu 165 170 175 Ile Lys Val Ser Gly Asn Ser Phe Val Gly Phe Ile Arg Asp Glu Tyr 180 185 190 Thr Thr Leu Pro Glu Asp Ser Asn Arg Pro Leu Phe Val Tyr Leu Asn 195 200 205 Ile Lys Trp Lys Tyr Lys Asn Thr Glu Asp Ser Phe Gly Thr Asn Pro 210 215 220 Glu Asn Tyr Val Ala Ala Glu Gln Ile Arg Asp Ile Ala Thr Ser Val 225 230 235 240 Phe His Glu Thr Glu Thr Leu Ser Ile Gln His Leu Ile Tyr Leu Ile 245 250 255 Gly Arg Arg Ile Leu Glu Arg Phe Pro Gln Leu Gln Glu Val Tyr Phe 260 265 270 Glu Ser Gln Asn His Thr Trp Asp Lys Ile Val Glu Glu Ile Pro Glu 275 280 285 Ser Glu Gly Lys Val Tyr Thr Glu Pro Arg Pro Pro Tyr Gly Phe Gln 290 295 300 Cys Phe Thr Val Thr Gln Glu Asp Leu Pro His Glu Asn Ile Leu Met 305 310 315 320 Phe Ser Asp Glu Pro Asp His Lys Gly Ala Leu Lys Pro Gly 325 330

<210> 5 <211> 29 <212> DNA <213> Artificial sequence <220> <223> the sequence of designed polynucleotide, described in example No. 1 <400> 5 ggtcatcacc atcaccatca ctgactgca 21

<210> 6 <211> 25 <212> DNA <213> Artificial sequence <220> <223> the sequence of designed polynucleotide, described in example No. 1 <400> 6 gtcagtgatg gtgatggtga tgacc 25

<210> 7 <211> 1101 <212> DNA <213> Bacillus sp. 109 Met Thr Lys His Lys Glu Arg Val Met Tyr Tyr Gly Lys Gly 1 5 10 gac gta ttt gct tat cgc acc tat tta aaa cca ctt act gga gtt aga 157 Asp Val Phe Ala Tyr Arg Thr Tyr Leu Lys Pro Leu Thr Gly Val Arg 15 20 25 30 acg att cct gaa tct cca ttt tcc ggt cga gat cat att ctt ttt gga 205 Thr Ile Pro Glu Ser Pro Phe Ser Gly Arg Asp His Ile Leu Phe Gly 35 40 45 gta aat gta aaa atc tca gta gga gga aca aaa ttg ctg acc tcc ttt 253 Val Asn Val Lys Ile Ser Val Gly Gly Thr Lys Leu Leu Thr Ser Phe 50 55 60 acg aaa ggg gat aac agc tta gtc gtt gca aca gac tcg atg aaa aac 301 Thr Lys Gly Asp Asn Ser Leu Val Val Ala Thr Asp Ser Met Lys Asn 65 70 75 ttt ata caa aaa cat tta gct agt tat aca gga aca acg ata gaa ggt 349 Phe Ile Gln Lys His Leu Ala Ser Tyr Thr Gly Thr Thr Ile Glu Gly 80 85 90 ttt tta gaa tat gta gct act tct ttt ttg aag aaa tat tct cat att 397 Phe Leu Glu Tyr Val Ala Thr Ser Phe Leu Lys Lys Tyr Ser His Ile 95 100 105 110 gaa aag att tcg ttg ata gga gag gaa att ccc ttt gaa aca act ttt 445 Glu Lys Ile Ser Leu Ile Gly Glu Glu Ile Pro Phe Glu Thr Thr Phe 115 120 125 gca gta aag aat gga aat aga gca gct agt gag cta gta ttt aaa aaa 49 3 Ala Val Lys Asn Gly Asn Arg Ala Ala Ser Glu Leu Val Phe Lys Lys 130 135 140 tca cga aat gaa tat gcc acc gct tat ttg aat atg gtt cgt aat gaa 541 Ser Arg Asn Glu Tyr Ala Thr Ala Tyr Leu Asn Met Val Arg Asn Glu 145 150 155 gat aac acc cta aac att act gaa caa caa agc gga ctt gct ggt ctt 589 Asp Asn Thr Leu Asn Ile Thr Glu Gln Gln Ser Gly Leu Ala Gly Leu 160 165 170 caa tta ata aaa gtc agc gga aat tcc ttt gtc ggt ttt att cgt gac 637 Gln Leu Ile Val Ser Gly Asn Ser Phe Val Gly Phe Ile Arg Asp 175 180 185 190 gaa tac aca act ctt cca gag gat tca aac cgc cct cta ttt gtt tac 685 Glu Tyr Thr Thr Thr Leu Pro Glu Asp Ser Asn Arg Pro Leu Phe Val Tyr 195 200 205 tta aac atc aaa tgg aag tac aaa aac acg gaa gac tca ttt gga acg 733 Leu Asn Ile Lys Trp Lys Tyr Lys Asn Thr Glu Asp Ser Phe Gly Thr 210 215 220 aat cca gaa aat tat gtt gca gct gaa caa att cgc atc gcc act 781 Asn Pro Glu Asn Tyr Val Ala Ala Glu Gln Ile Arg Asp Ile Ala Thr 225 230 235 tcc gta ttt cat gaa acc gag acg ctt tcc atc caa cat tta att tat 829 Ser Val Phe His Glu Thr Glu Thr Leu Ser Ile Gln His Leu Ile Tyr 240 245 250 tta atc ggc cgc aga ata tta gaa aga ttc cct caa ctt caa gaa gtt 877 Leu Ile Gly Arg Arg Ile Leu Glu Arg Phe Pro Gln Leu Gln Glu Val 255 260 265 270 270 tac ttcga caa aat cat aca tgg gat aaa ata gtg gag gaa att 925 Tyr Phe Glu Ser Gln Asn His Thr Trp Asp Lys Ile Val Glu Glu Ile 275 280 285 cct gaa tca gaa ggg aaa gta tat aca gaa ccg cga ccg cca tat g Glu Ser Glu Gly Lys Val Tyr Thr Glu Pro Arg Pro Pro Tyr Gly 290 295 300 ttt caa tgc ttt act gtc acc caa gaa gac ttg cca cac gaa aac att 1021 Phe Gln Cys Phe Thr Val Thr Gln Glu Asp Leu Pro His Glu Asn Ile 305 310 315 ctt atg ttc tct gat gaa ccc gat cat aaa gga gca ctt aaa ccc ggt 1069 Leu Met Phe Ser Asp Glu Pro Asp His Lys Gly Ala Leu Lys Pro Gly 320 325 330 cat cac cat cac cat cac tgactgcaga tatc 1101 His His His His His His 335 340

<210> 8 <211> 340 <212> PRT <213> Bacillus sp.TB-90 <400> 8 Met Thr Lys His Lys Glu Arg Val Met Tyr Tyr Gly Lys Gly Asp Val 1 5 10 15 Phe Ala Tyr Arg Thr Tyr Leu Lys Pro Leu Thr Gly Val Arg Thr Ile 20 25 30 Pro Glu Ser Pro Phe Ser Gly Arg Asp His Ile Leu Phe Gly Val Asn 35 40 45 Val Lys Ile Ser Val Gly Gly Thr Lys Leu Leu Thr Ser Phe Thr Lys 50 55 60 Gly Asp Asn Ser Leu Val Val Ala Thr Asp Ser Met Lys Asn Phe Ile 65 70 75 80 Gln Lys His Leu Ala Ser Tyr Thr Gly Thr Thr Ile Glu Gly Phe Leu 85 90 95 Glu Tyr Val Ala Thr Ser Phe Leu Lys Lys Tyr Ser His Ile Glu Lys 100 105 110 Ile Ser Leu Ile Gly Glu Glu Ile Pro Phe Glu Thr Thr Phe Ala Val 115 120 125 Lys Asn Gly Asn Arg Ala Ala Ser Glu Leu Val Phe Lys Lys Ser Arg 130 135 140 Asn Glu Tyr Ala Thr Ala Tyr Leu Asn Met Val Arg Asn Glu Asp Asn 145 150 155 160 Thr Leu Asn Ile Thr Glu Gln Gln Ser Gly Leu Ala Gly Leu Gln Leu 165 170 175 Ile Lys Val Ser Gly Asn Ser Phe Val Gly Phe Ile Arg Asp Glu Tyr 180 185 190 Thr Thr Leu Pro Glu Asp Ser Asn Arg Pro Leu Phe Val Tyr Leu Asn 195 200 205 Ile Lys Trp Lys Tyr Lys Asn Thr Glu Asp Ser Phe Gly Thr Asn Pro 210 215 220 Glu Asn Tyr Val Ala Ala Glu Gln Ile Arg Asp Ile Ala Thr Ser Val 225 230 235 240 Phe His Glu Thr Glu Thr Leu Ser Ile Gln His Leu Ile Tyr Leu Ile 245 250 255 Gly Arg Arg Ile Leu Glu Arg Phe Pro Gln Leu Gln Glu Val Tyr Phe 260 265 270 Glu Ser Gln Asn His Thr Trp Asp Lys Ile Val Glu Glu Ile Pro Glu 275 280 285 Ser Glu Gly Lys Val Tyr Thr Glu Pro Arg Pro Pro Tyr Gly Phe Gln 290 295 300 Cys Phe Thr Val Thr Gln Glu Asp Leu Pro His Glu Asn Ile Leu Met 305 310 315 320 Phe Ser Asp Glu Pro Asp His Lys Gly Ala Leu Lys Pro Gly His His 325 330 335 His His His His 340

[Brief description of the drawings]

FIG. 1 is a diagram showing the creation of a gene encoding a fusion protein.

FIG. 2 is a view showing the results of an SDS-polyacrylamide gel analyzing the fusion protein and the state of selective cleavage thereof.

FIG. 3 is a diagram showing a flow of high-throughput purification of a protein using affinity beads.

FIG. 4 is a view showing the results of SDS-polyacrylamide gel obtained by analyzing the fusion protein and the state of selective cleavage thereof.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B024 AA03 BA08 CA04 DA06 EA04 GA11 4B050 CC01 CC03 DD20 EE10 LL05 4B064 AG01 CA21 CB02 DA20 4H045 AA20 AA30 BA41 CA40 DA86 DA89 FA30

Claims (11)

[Claims] (1) a fusion protein comprising a tag and a protein,
A method of cleaving with a non-specific endoprotease. 2. In the step of cleaving the sequence of the linking portion or its peripheral portion without cutting the sequence of the protein portion to obtain the desired protein, the fusion protein in which the tag and the protein are linked is non-specific. A method for obtaining a useful protein from a fusion protein, characterized by using an endoprotease. 3. The method according to claim 2, wherein the amino acid sequence of the connecting portion is represented by the formula (1). X-Met-Phe-Ser- (Xaa) nY (1) wherein X represents a protein and Y represents a tag. Xaa means any amino acid residue. n is an integer of 0 to 50. ] 4. The method according to claim 2, wherein the amino acid sequence of the connecting portion is represented by the formula (2). X-Met-Phe-Ser-Asp-Glu-Pro-Asp-His-Lys-Gly-Ala-Leu-
Lys- (Xaa) nY (2) [wherein, X represents a protein and Y represents a tag. Xaa means any amino acid residue. n is an integer of 0 to 40. ] 5. The method according to claim 2, wherein the amino acid sequence of the connecting portion is represented by the formula (3). X-Met-Phe-Ser-Asp-Glu-Pro-Asp-His-Lys-Gly-Ala-Leu-
Lys-Y (3) [where X represents a protein and Y represents a tag. ] 6. The method according to claim 1, wherein the tag is an affinity peptide containing an adjacent histidine residue. 7. The method according to claim 1, wherein the protein is an enzyme. 8. The method according to claim 7, wherein the enzyme is uricase. 9. Uricase is Bacillus sp. TB90
9. The method according to claim 8, wherein the method is of origin. 10. The method according to claim 1, wherein the non-specific endoprotease is selected from proteinase K, trypsin, chymotrypsin and V8 protease. 11. The method according to claim 10, wherein the non-specific endoprotease is proteinase K.