JP2003038171A - Method for obtaining useful protein from fused protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for selectively fragmenting a fused protein to obtain a desired protein therefrom, and further to provide a method for obtaining the desired protein from the fused protein by enzymatically fragmenting the amino acid sequence at the linked site or the vicinity thereof. SOLUTION: This method for specifically obtaining the desired protein comprises a step of selectively fragmenting the sequence of the fused protein obtained by linking a peptide having a specific amino acid sequence with a protein having a specified molecular weight, at the linked site or the vicinity thereof. The method for obtaining the useful protein from the fused protein uses an endoprotease having nonspecificity or low specificity to the sequence in the step.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a selective cleavage method for obtaining a desired protein from a fusion protein. The present invention also relates to a method for obtaining a desired protein from a fusion protein, which is characterized by enzymatically cleaving the amino acid sequence of the connecting portion and its peripheral portion. INDUSTRIAL APPLICABILITY The present invention is useful for fusion proteins and the like that are difficult to selectively cleave by ordinary methods. Further, according to the present invention, it becomes possible to prepare a desired protein from a fusion protein easily at a very low cost.

[0002]

2. Description of the Related Art Advances in genetic engineering have made it possible to artificially amplify a target protein by analyzing, at the gene level, a protein that has been purified from a natural product. Thioredoxin invented after that (Tokuhyo 5-507209)
And glutathione-S-transferase (GST)
By applying a DNA sequence in which (Table 1-503441) and the like are fused, it becomes possible to express even a protein that is difficult to express originally, and the technique for expressing a fusion protein has become widely used. It 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 this purpose, various peptides (tags) having a specific amino acid sequence have been developed for use in the preparation of fusion proteins. As a result, an affinity peptide (His-tag) containing adjacent histidine residues (JP-A-63-2).
51095) and maltose binding protein (JP-A 1-20
094) and the like and a desired protein are fused via a connecting portion, and a technique for easily purifying the produced fusion protein using an affinity chromatography technique or the like has been established. However, in order to obtain the 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 physiological activities as the desired protein, the fusion protein can be directly used for the application, but this is rare. Many fusion proteins are affected by the fused peptides, and their properties and physiological activities are considerably changed. As a method for removing the fused peptide, an enzymatic cleavage method or a chemical reaction method can be considered.However, in order to obtain the desired protein in a stable state, an enzymatic cleavage method is generally adopted. It Enzymatic cleavage method is a peptide fused with a sequence-specific protease recognition amino acid sequence that has been inserted in advance at the junction between the desired protein and the fused peptide, and the corresponding sequence-specific protease is allowed to act. Is a method of removing. A sequence-specific protease is an enzyme capable of recognizing and cleaving only a specific amino acid sequence, and there is no fear of cleaving a desired protein. However, sequence-specific proteases are extremely expensive, and it is not practical to use this method to create a practical enzyme, and a new method that is inexpensive and has the same effect and convenience as sequence-specific proteases is desired. It was rare.

[0003]

For these reasons,
There has been a need for an inexpensive and convenient method for selectively cleaving the fusion protein. That is, an object of the present invention is to provide a method for selectively cleaving a fusion protein by a simple method that is inexpensive and convenient.

[0004]

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 arrived at the present invention. More specifically, the inventors have found that the use of a non-specific endoprotease makes it possible to obtain a useful protein from a fusion protein having a specific amino acid sequence in the linking portion, and arrived at the present invention. Until now, it has been considered that non-specific endoprotease does not recognize a specific sequence and cleaves the protein at a plurality of sites, and thus it has been considered impossible to use it for a method of selectively cleaving a fusion protein. However, we conducted various studies and made it possible to obtain a useful protein from a fusion protein having a specific amino acid sequence in the connecting portion by using a non-specific endoprotease.

That is, the present invention has the following configuration. [1] A method of cleaving a fusion protein in which a tag and a protein are linked with a non-specific endoprotease. [2] A non-specific endoprotease for a fusion protein in which a tag and a protein are ligated, in which a desired protein is obtained by cleaving the sequence of the ligation part or its peripheral part without cleaving the sequence of the protein part. A method for obtaining a useful protein from a fusion protein, which comprises using [3] The method according to [2], wherein the sequence easily recognized by the non-specific endoprotease is represented by the formula (1). X-Met-Phe-Ser- (Xaa) nY (1) [In the formula, X means a protein and Y means a tag. Xaa means any amino acid residue. n is an integer of 0 to 50. [4] The method according to [2], wherein the sequence that is easily recognized by the non-specific endoprotease is represented by the formula (2). X-Met-Phe-Ser-Asp-Glu-Pro-Asp-His-Lys-Gly-Ala-Leu-Lys- (Xaa) nY (2) [where X is a protein and Y is a tag] To do. Xaa means any amino acid residue. n is an integer of 0-40. [5] The method according to [2], wherein the sequence easily recognized by the non-specific endoprotease is represented by the formula (3). X-Met-Phe-Ser-Asp-Glu-Pro-Asp-His-Lys-Gly-Ala-Leu-Lys-Y (3) [wherein 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 adjacent histidine residues. [7] The method according to [1] to [6], wherein the protein is an amine oxidase. [8] The method according to [1] to [7], wherein the non-specific endoprotease is selected from proteinase K, trypsin, chymotrypsin, and V8 protease. [9] Proteinase K is a non-specific endoprotease
The method according to [8], wherein

In the present invention, the non-specific endoprotease refers to a protease which does not recognize a specific sequence and can cleave a protein at a plurality of sites. Specifically, when it reacts with a protein, it does not recognize a specific sequence and cleaves the protein at a plurality of locations, but when it reacts with the fusion protein of the protein according to the present invention, it does not cleave the sequence of the protein part, 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) yield of 60% or more when 1 g / L protease is reacted with 3 g / L protein in 20 mM Tris-HCl buffer at pH 7.5 at 37 ° C. for 30 minutes, (B) A protease having the property that the yield of the desired protein exceeds 40% when it is reacted with the fusion protein of the protein according to the present invention under the same conditions. Preferably, the protease has a property such that the yield of the protein in (a) is 80% or more, but the yield of the desired protein according to the present invention in (b) exceeds 70%, more preferably , (A), the yield of the protein is 95% or more, while in (b), the yield of the desired protein of the present invention is 90% or more. The yield can be determined by measuring the activity in the case of an enzyme or the like, or by an immunoassay or the like. That is, the yield can be determined by determining the activity or the ratio of immunoassay values before and after the treatment with protease.

Alternatively, for example, 1 g / L of protease is added to 3 g / L in 20 mM Tris-HCl buffer of pH 7.5.
It is a protease having such a property that, when it is reacted with L protein at 37 ° C. for 30 minutes, the ratio of the largest amount of degradation products to the total degradation products does not exceed 40%. Preferred is a protease having properties such that the ratio of the highest amount of degradation products to the whole does not exceed 20%. More preferably, it is a protease having properties such that the ratio of the highest amount of degradation products to the whole does not exceed 10%. The ratio of degradation products is, for example, SDS-PAG.
It can be calculated from data such as E. That is, the ratio can be determined by applying the SDS-PAGE result of the reaction solution to a one-dimensional image analyzer and a densitometer and calculating the band concentration of each degradation product as a numerical value.

The property of the target protein in the method of the present invention is not particularly limited, but a single protein having a specific molecular weight is preferable.

The method for selectively cleaving the 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 protein of interest are linked. (2) Synthesis of fusion protein. (3) Preparation of fusion protein. (4) Enzymatic degradation of the sequence of the fusion protein connecting region or its peripheral region. (5) Preparation of desired protein. However, even if the steps (4) to (5) are put together, substantially the same result will be obtained.

In the present invention, the means for synthesizing the fusion protein used is not particularly limited. As the method, conventionally used genetic engineering methods, modified methods thereof, and protein engineering methods can be used. In particular,
The tag and the protein can be linked at the gene level, and the produced fusion protein gene can be expressed in a recombinant bacterium using a gene recombination technique and synthesized. Furthermore, it is also possible to increase the purity of the fusion protein and obtain a substantially single protein preparation by using a culture / purification technique. A general method for obtaining a target protein using a microbial gene manipulation system 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 cleaved using ultrasonic disruption, restriction enzyme treatment, or the like.
A recombinant expression vector is constructed by ligating and closing a linear expression vector at the blunt ends or cohesive ends of both DNAs with DNA ligase or the like. The recombinant vector thus obtained is transferred into a replicable host microorganism and then screened using the expression of the vector marker and the enzyme activity as an index, and a microorganism carrying the recombinant vector containing the gene encoding the desired protein. To get Then, the microorganism may be cultured, the recombinant vector may be separated and purified from the cultured cells, and the gene of the desired protein may be collected from the recombinant vector.

The chromosomal DNA is specifically collected as follows. That is, the gene-containing lysate can be prepared by collecting the culture obtained by stirring and culturing the donor organism for 1 to 3 days by centrifugation, and then lysing the culture. As a lysing method, for example, treatment is performed with a lysing enzyme such as lysozyme or β-glucanase, and if necessary, a protease or other enzyme and a surfactant such as sodium lauryl sulfate (SDS) are used together, and further freeze-thawing It may be combined with a physical crushing method such as or French press treatment. In order to separate and purify DNA from the lysate thus obtained, a conventional method may be used, for example, deproteinization treatment by phenol treatment or protease treatment, ribonuclease treatment, alcohol precipitation treatment and the like are appropriately combined. You can The method of cleaving the separated / purified DNA can be carried out, for example, by ultrasonic treatment or restriction enzyme treatment. Type II restriction enzymes that act on specific nucleotide sequences are preferred.

Suitable vectors are those constructed for gene recombination from phages or plasmids capable of autonomous growth in host microorganisms. Examples of phages include Escherichia col
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
D-M1 (Journal of Fermentation and Bioenzineerin
g, Vol.76, 265-269 (1993)) can be used. Such a vector can be cleaved with the above-mentioned restriction enzyme used for the cleavage of the stained DNA to obtain a vector fragment, but it is not always necessary to use the same restriction enzyme as that used for the cleavage of the chromosomal DNA. . The method for ligating the chromosomal DNA fragment and the vector DNA fragment is known in the art.
A method using NA ligase may be used, for example, after the cohesive ends of the chromosomal DNA fragment and the cohesive ends of the vector fragment are annealed, a recombinant vector of the chromosomal DNA fragment and the vector DNA fragment is prepared by using an appropriate DNA ligase. To do. If necessary, after annealing, it can be transferred into a host microorganism and utilized in vivo DNA ligase to prepare a recombinant vector.

The host microorganism may be any as long as the recombinant vector is stable, capable of autonomous growth, 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 of transferring the recombinant vector into the host microorganism, for example, when the host microorganism is Escherichia coli, the competent cell method by calcium treatment or the electroporation method can be used.
The thus obtained transformant microorganism can stably produce a large amount of protein by culturing in a nutrient medium. The selection as to whether or not the target recombinant vector has been transferred into the host microorganism can be carried out by searching for a microorganism that expresses the drug resistance marker of the vector carrying the target DNA. The recombinant vector obtained by the above method can be easily taken out from a transformed microorganism and transferred to another microorganism. In addition, DN, which is a gene encoding a protein by restriction enzyme or PCR
It is also easy to collect A, combine it with another vector fragment, and transfer it to a host microorganism.

Regarding the culture form of the host microorganism which is a transformant, the culture conditions may be selected in consideration of the nutritional physiological properties of the host. Usually, in most cases, liquid culture is carried out, but industrially aeration stirring is carried out. It is advantageous to carry out culturing. As the nutrient source of the medium, those usually used for culturing microorganisms can be widely used. The carbon source may be any assimilable carbon compound, and for example, glucose, sucrose, lactose, maltose, fructose, molasses, pyruvic acid, etc. are used. Any available nitrogen compound may be used as the nitrogen source, and for example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean meal alkali extract, etc. are used. In addition, salts such as phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese and zinc, specific amino acids, specific vitamins and the like are used as necessary. The culturing temperature may be appropriately changed within the range in which the bacterium grows and produces the desired protein, but in the case of Escherichia coli, it is preferably about 20 to 42 ° C. Although the culturing time varies slightly depending on the conditions, it may be completed at a suitable time in consideration of the time when the desired protein reaches the maximum yield, and it is usually about 6 to 48 hours. Medium pH
Can be appropriately changed within the range in which the bacterium grows and produces the desired protein, but the pH is 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.
Generally, when the target protein is present in the culture solution according to a conventional method, it is used after separating the solution containing the target protein from the microbial cells by filtration, centrifugation, or the like. When the target protein is present in the microbial cells, the microbial cells are collected from the obtained culture by means such as filtration or centrifugation, and then the microbial cells are destroyed by a mechanical method or an enzymatic method such as lysozyme. And EDT if necessary
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 desired protein thus obtained is concentrated under reduced pressure, membrane concentration, salting out with ammonium sulfate, sodium sulfate or the like, or a hydrophilic organic solvent such as methanol, ethanol or acetone. It may be precipitated by the fractional precipitation method according to. Further, heat treatment and isoelectric point treatment are also effective refining means. Then, gel filtration using an adsorbent or a gel filter, adsorption chromatography, ion exchange chromatography, or affinity chromatography can be performed to obtain the purified target protein. For example, Sephadex G-25 (Pharmacia
Biotech) gel filtration, DEAE Sepharose CL
-6B (Pharmacia Biotech), octyl sepharose CL6-B (Pharmacia Biotech) column chromatography can be separated and purified to obtain a purified enzyme preparation. This purified enzyme preparation has been purified by electrophoresis (SDS-PAGE) so as to show an almost single band.

Alternatively, the fusion protein may be synthesized using a cell-free protein synthesis method. With the progress of biotechnology in recent years, the utility value of protein synthesis using the cell-free protein synthesis method is rapidly attracting attention. Cell-free protein synthesis takes out components related to protein synthesis in the living body to the outside of the body, and extracts mRNA that encodes the target protein.
It is a means to synthesize proteins artificially by adding energy sources such as ATP and GTP. Escherichia coli, reticulocytes, and wheat germ are often used as the biomaterials, and various preparation methods and utilization methods have been studied so far. The advantages of cell-free protein synthesis method over protein synthesis methods using recombinant microorganisms and cultured cells are:
1) Proteins that negatively act on the body (excluding those that affect the translation system) are relatively easy to synthesize. 2) Conditions can be easily determined (minimum when using microorganisms). It usually takes about a month) 3)
It can be mentioned that non-natural amino acids can be used.

In the present invention, the origin of the sequence-nonspecific protease used for enzymatically degrading the sequence of the connecting portion of the fusion protein or its peripheral portion is not particularly limited, but belongs to serine protease, thiol protease and the like. It is preferable to use protease, specifically proteinase K, trypsin, chymotrypsin, subtilisin, V8 protease and the like. The condition of the decomposition reaction by protease depends on the enzymatic properties of each protease, but generally, the pH of the reaction solution is fixed at 10 to 90 by fixing the pH of the reaction solution with a buffer solution such as GOOD buffer or phosphate buffer.
The reaction is performed at 5 ° C. for 5 minutes to 24 hours. For details, for example,
Neutral protease is weakly acidic to weakly alkaline Tris-HCl
The reaction is carried out in the buffer at 20 to 80 ° C. for 10 minutes to 16 hours. Alternatively, use alkaline protease at pH 8-11.
30 to 16 minutes at 10 to 60 ° C. in GOOD buffer of
React for hours.

The fusion protein used in the present invention may be used as it is after its synthesis after synthesis, or may be purified. Preferably, it is purified to high purity using ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration, hydroxyapatite, or isoelectric focusing chromatography. More preferably, thioredoxin, GST, Hi
It is also possible to carry out affinity purification 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 any protein whose existence can be confirmed in any form may be used.
Enzymes include uricase, catalase, amine oxidase, glucose oxidase, amino acid dehydrogenase, DNA polymerase, DNA ligase, restriction enzyme,
DNA as a protein whose existence can be confirmed, such as protein kinase, protease, lipase, and amylase
Examples include binding proteins, receptor proteins, chaperonins, cytokines, hormone-like proteins and the like.

One aspect of the present invention is a method for easily and inexpensively obtaining a desired protein from a fusion protein. Another aspect of the present invention is a reagent system for research on protein purification / 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 by 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 the examples of the present invention, but the present invention is not limited thereto. Example 1 Preparation of Gene Encoding Fusion Protein As a desired protein, Bacillus sp. TB, which is a practical enzyme used in a clinical test drug for measuring uric acid
Uricase from 90 strains (urate oxidase, EC 1.7.3.3) was taken up. As a peptide having a specific amino acid sequence, an affinity peptide containing adjacent histidine residues, specifically 6xHis-t in which 6 histidine residues are arranged.
Taking ag, a gene encoding a fusion protein in which this was bound to the carboxyl terminus of uricase via a linking portion was prepared by the following procedure. The flow of creation is also shown in FIG. Plasmid pUOD containing uricase gene (publication of JP-A-2-53488) (Journ
al of Biochemistry, Vol.119, 80-84 (1996)) (Bioindustry, Vol.13, 20-28 (1996)) as a template, and polymerase chain reaction (PCR) under the following composition and conditions. The uricase gene for preparing the fusion protein gene was prepared. 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 x10 buffer (Toyobo) 5 μl 2 mM dNTP (Toyobo) 5 μl 25 mM MgSO4 (Toyobo) 4 μl Total solution 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 was described in SEQ ID NO: 3 in the sequence listing, and the amino acid sequence of uricase was described in SEQ ID NO: 4 in the sequence listing. The obtained uricase gene is a PCR product purification kit (MagExtractor-PCR
& Gel Clean Up- (manufactured by Toyobo) according to the protocol, and cut with restriction enzymes EcoRI and PstI.
And the expression vector pKK223-3 (manufactured by Amersham Pharmacia) cleaved with PstI and T4 DNA ligase were used for ligation. The resulting uricase gene expression plasmid was named pUODSP1 (FIG. 1). pUO
DSP1 was cleaved with restriction enzymes SmaI and PstI to cleave the carboxyl terminal side of the uricase gene. Synthetic oligonucleotides having the sequences of SEQ ID NOs: 5 and 6 in the sequence listing were ligated to this using T4 DNA ligase, and finally 6xHis-tag in which 6 histidine residues were arranged had a ligation portion at the carboxyl terminal of uricase. An expression plasmid, pUODSP1-HT, containing a gene encoding the fusion protein bound via the plasmid could be constructed (FIG. 1). The sequence of the gene encoding the prepared fusion protein is described in SEQ ID NO: 7 in the sequence listing, and the amino acid sequence of the fusion protein is described in SEQ ID NO: 8 in the sequence listing.

Example 2 Preparation of Fusion Protein A competent cell of Escherichia coli JM109 (Competent High (manufactured by Toyobo)) was transformed with pUODSP1-HT to prepare a fusion protein-producing recombinant bacterium JM109 / pUODSP.
1-HT was prepared. 50 ml of this, TB-brot
Inoculate into a 500 ml volumetric flask containing h and incubate at 37 ° C for 1
Cultured for 7 hours. The bacterial cells were collected from the culture solution by centrifugation, and buffer A (20 mM Tris-HCl (pH 7.
5), after suspending with 350 mM NaCl), the protein was extracted by ultrasonication. Crush the supernatant with nickel chelate
Pass through column chromatography (HiTrap-Chelating- (manufactured by Amersham Pharmacia)) and follow buffer B (20 mM Tris-H) according to the manufacturer's protocol.
Cl (pH 7.5), 350 mM NaCl, 500 m
(M imidazole). Furthermore, the eluate is Se
It is passed through a phasex G-25 desalting / column chromatography (Hitrap-Desalting- (manufactured by Amersham Pharmacia)), eluted with buffer C (20 mM Tris-HCl (pH 7.5)) according to the manufacturer's protocol, and the buffer is replaced. , A purified preparation of the fusion protein was prepared. The analysis results of the disrupted supernatant and the purified protein by SDS-polyacrylamide gel are shown in FIG.

Example 3 Treatment with non-specific protease and preparation of desired protein Purified preparation of fusion protein was prepared by using proteinase K (Nacalai Tesque) 20 mM Tris-HCl (pH 7.
5) Treated with solution. 50 μl fusion protein: about 2 mg
/ Μl proteinase K: about 0.1 to 1 μg / m
It was added at a final concentration of 1 and treated at 37 ° C. for 30 minutes. The reaction was terminated by adding PMSF (phenylmethylsulfonyl fluoride (manufactured by Nacalai Tesque)), which is an inhibitor of proteinase K, at 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, which was the desired protein, could be obtained. The analysis results of the proteinase K-treated product of the fusion protein by SDS-polyacrylamide gel are shown in FIG. The connecting portion of the fusion protein is cleaved by the enzymatic degradation of proteinase K to give 6xH.
The analysis results revealed that the uricase was being prepared by removing the is-tag peptide. Cleavage of the junction of the fusion protein by a non-specific protease
It was also confirmed by mass spectrometry by LDI-TOFF-MASS. It is speculated that the selective cleavage of the fusion protein by a non-specific protease is due to the structural characteristics of the ligation part or the ligation part and its peripheral part, but at this time, the detailed basic mechanism has not been clarified, and its application. Only its utility in terms of aspects is known. Therefore, X-Met-Phe-Ser- (Xaa) nY (1) is characterized in that the amino acid sequence of the linking portion is represented by formula (1) [wherein X represents a protein of 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. Alternatively, 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) [In the formula, X means a protein having a specific molecular weight, and Y means a peptide having a specific amino acid sequence. Xaa means any amino acid residue. n is an integer of 0-40. Alternatively, 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) [In the formula, X means a protein having a specific molecular weight, and Y means a peptide having a specific amino acid sequence. The information as a novel method is disclosed as an example.

Example 4 Affinity of Fusion Protein
Preparation of desired protein by binding to beads and treatment with non-specific protease I By using the method of the present patent, it is possible to bind a fusion protein to affinity beads, which has been substantially difficult in terms of cost, by sequence selection. A method of obtaining a desired protein by cleavage becomes possible. By enabling this technique, high-throughput purification of proteins can be easily carried out. An example of the flow of high throughput protein purification by the method of this patent is shown in FIG. Similar to 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 bacterial cells are collected from the culture solution by centrifugation to collect the buffer A.
After suspending in, the protein was extracted by ultrasonication. Disintegrate liquid with nickel chelate / magnetic beads (MagExtractor-His
-tag- (manufactured by Toyobo)), and the beads were washed with the attached buffer according to the manufacturer's protocol. By this operation, magnetic beads to which the fusion protein is adsorbed are prepared. The magnetic beads were used as proteinase K (manufactured by Nacalai Tesque, Inc.) 20 mM Tris-HCl (pH 7.
5) Treatment with the solution 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, and the mixture was treated 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, which was the desired protein, could be obtained. Proteinase K for magnetic beads
The analysis result of the treated product by SDS-polyacrylamide gel is shown in FIG. The connecting portion of the fusion protein is cleaved by the enzymatic decomposition action of proteinase K to give 6xHi.
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 fusion protein junction by a non-specific protease was also confirmed by mass spectrometry by MALDI-TOFF-MASS. Example 5 Preparation of desired protein by binding of fusion protein to affinity bead and treatment with non-specific protease II By using the method of this patent, high-throughput purification of amine oxidase can be carried out easily. The sequence of the gene encoding the amine oxidase fusion protein is shown in SEQ ID NO: 9 in the sequence listing, and the amino acid sequence of the fusion protein is shown in SEQ ID NO: 10 in the sequence listing. In the same manner as in Example 4, the recombinant protein producing recombinant protein of amine oxidase was treated with TB-brot.
The cells were inoculated into h and cultured at 37 ° C. for 17 hours. The cells were collected from the culture solution by centrifugation, suspended in buffer A, and then sonicated to extract the protein. The disrupted solution was adsorbed on nickel chelate / magnetic beads (MagExtractor-His-tag- (Toyobo)), and the beads were washed with the attached buffer according to the manufacturer's protocol. By this operation, magnetic beads to which the fusion protein is adsorbed are prepared. This magnetic bead was used as Proteinase K (manufactured by Nacalai Tesque) 20
Treatment with a mM Tris-HCl (pH 7.5) solution eluted the desired amine oxidase. Magnetic beads 2
About 1 to 4 μg of proteinase K solution for 0 μl volume
100 μl was added at a concentration of / ml and treated 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,
The desired amine oxidase could be obtained. Since magnetic beads are used in this example, solid-liquid separation can be performed very easily by using a magnet without performing centrifugation. The solid-liquid separation system for magnetic beads using a magnet is easy to automate, and in fact, an automation device has already been developed and sold (MFX-2000, MFX-6000, MF
X-9600 (made by Toyobo)). Therefore, by combining these apparatuses with the method of the present patent, high-throughput purification of proteins can be easily realized.

[0028]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to selectively cleave a fusion protein with a non-specific protease, which has been difficult with conventional techniques. This makes it possible to easily and inexpensively prepare a desired protein from the fusion protein.
Further, by taking advantage of the cost advantage of this 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> 01-0505 <141> 2001-07-03 <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, described in example No.1 <400> 1   aaacagaatt cgagctcgcg c 21

[0030] <210> 2 <211> 41 <212> DNA <213> Artificial sequence <220> <223> the sequence of designed polynucleotide for PCR primer, described in example No.1 <400> 2   gatatctgca gtcacccggg tttaagtgct cctttatgat c 41

[0031] <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 gtg atg tat tat gga aaa ggt 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 Lys 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 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 gac 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 tac ttc gaa tct 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 gga 973 Pro 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 ggg 1069 Leu Met Phe Ser Asp Glu Pro Asp His Lys Gly Ala Leu Lys Pro Gly     320 325 330 tgactgcaga tatc 1083

[0032] <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

[0033] <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

[0034] <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

[0035] <210> 7 <211> 1101 <212> DNA <213> Bacillus sp.TB-90 <400> 7 aaacagaatt cgagctcgcg caatgtttcg tttgcaagaa atatttcgtg aaggagagaa 60 taattcg atg acc aaa cac aaa gaa aga gtg atg tat tat gga aaa ggt 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 Lys 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 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 gac 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 tac ttc gaa tct 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 gga 973 Pro 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

[0036] <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

[0037] <210> 9 <211> 1236 <212> DNA <213> Arthrobacter sp. TE1826 <400> 9 atg agt att aaa aaa gat tat gat gta att gtg gtt ggc gct ggt tcc 48 Met Ser Ile Lys Lys Asp Tyr Asp Val Ile Val Val Gly Ala Gly Ser   1 5 10 15 atg gga atg gca gct ggg tac tat ctg tct aaa caa ggt gtt aaa aca 96 Met Gly Met Ala Ala Gly Tyr Tyr Leu Ser Lys Gln Gly Val Lys Thr              20 25 30 cta ttg gta gat tca ttt gat cct ccc cat aca aat ggc agc cat cat 144 Leu Leu Val Asp Ser Phe Asp Pro Pro His Thr Asn Gly Ser His His          35 40 45 ggc gat aca cgg atc att cgt cac gca tat ggc gaa gga aga gag tat 192 Gly Asp Thr Arg Ile Ile Arg His Ala Tyr Gly Glu Gly Arg Glu Tyr      50 55 60 gta ccg ttt gcc ttg aga gca caa gag tta tgg tat gaa tta gaa aag 240 Val Pro Phe Ala Leu Arg Ala Gln Glu Leu Trp Tyr Glu Leu Glu Lys  65 70 75 80 gag act cat cat aaa ata ttt aca aaa aca ggt gta ctc gtt ttt ggt 288 Glu Thr His His Lys Ile Phe Thr Lys Thr Gly Val Leu Val Phe Gly                  85 90 95 cct aaa gga gaa gct cct ttc gtt gcc gaa aca atg gaa gcc gca aag 336 Pro Lys Gly Glu Ala Pro Phe Val Ala Glu Thr Met Glu Ala Ala Lys             100 105 110 gaa cat tca tta gat gtt gat tta cta gaa gga agt gaa ata aat aag 384 Glu His Ser Leu Asp Val Asp Leu Leu Glu Gly Ser Glu Ile Asn Lys         115 120 125 cgt tgg cca ggt gta acg gtt cct gag aat tat aat gct att ttt gaa 432 Arg Trp Pro Gly Val Thr Val Pro Glu Asn Tyr Asn Ala Ile Phe Glu     130 135 140 aaa aat tct ggt gtc tta ttt agt gaa aat tgt att cgc gct tac cgt 480 Lys Asn Ser Gly Val Leu Phe Ser Glu Asn Cys Ile Arg Ala Tyr Arg 145 150 155 160 gaa ttg gcg gaa gca aat ggt gcg aaa gtt cta acg tac aca ccc gtt 528 Glu Leu Ala Glu Ala Asn Gly Ala Lys Val Leu Thr Tyr Thr Pro Val                 165 170 175 gaa gat ttc gag att gcc gag gac ttc gtc aaa atc caa acc gcc tat 576 Glu Asp Phe Glu Ile Ala Glu Asp Phe Val Lys Ile Gln Thr Ala Tyr             180 185 190 ggc tcc ttt aca gcc agt aaa tta att gtt agc atg ggc gct tgg aat 624 Gly Ser Phe Thr Ala Ser Lys Leu Ile Val Ser Met Gly Ala Trp Asn         195 200 205 agc aaa ctg cta tca aaa tta aat att gaa atc cca ttg cag cca tac 672 Ser Lys Leu Leu Ser Lys Leu Asn Ile Glu Ile Pro Leu Gln Pro Tyr     210 215 220 cgt caa gtt gtc gga ttc ttc gaa tgt gat gaa aaa aaa tat agc aat 720 Arg Gln Val Val Gly Phe Phe Glu Cys Asp Glu Lys Lys Tyr Ser Asn 225 230 235 240 aca cat ggt tat ccg gcg ttc atg gtc gaa gtc cca act ggc atc tat 768 Thr His Gly Tyr Pro Ala Phe Met Val Glu Val Pro Thr Gly Ile Tyr                 245 250 255 tac gga ttt cca agc ttc ggc ggc tgc ggc ttg aaa ata ggc tat cat 816 Tyr Gly Phe Pro Ser Phe Gly Gly Cys Gly Leu Lys Ile Gly Tyr His             260 265 270 acg tat ggt caa aaa atc gat cca gat acg att aat cgt gaa ttt ggt 864 Thr Tyr Gly Gln Lys Ile Asp Pro Asp Thr Ile Asn Arg Glu Phe Gly         275 280 285 att tac ccg gag gat gaa ggg aat att cgc aaa ttc ctg gaa aca tat 912 Ile Tyr Pro Glu Asp Glu Gly Asn Ile Arg Lys Phe Leu Glu Thr Tyr     290 295 300 atg ccg gga gca acc ggc gaa tta aaa agt ggg gca gtt tgc atg tac 960 Met Pro Gly Ala Thr Gly Glu Leu Lys Ser Gly Ala Val Cys Met Tyr 305 310 315 320 aca aaa aca cct gat gag cat ttc gtg att gat tta cat cct caa ttc 1008 Thr Lys Thr Pro Asp Glu His Phe Val Ile Asp Leu His Pro Gln Phe                 325 330 335 tcg aat gtc gcg att gca gcc gga ttc tcc gga cat ggg ttt aaa ttc 1056 Ser Asn Val Ala Ile Ala Ala Gly Phe Ser Gly His Gly Phe Lys Phe             340 345 350 tca agc gta gtt ggt gaa aca tta agt caa tta gct gta acc ggt aaa 1104 Ser Ser Val Val Gly Glu Thr Leu Ser Gln Leu Ala Val Thr Gly Lys         355 360 365 aca gaa cac gat att tcc atc ttt tca atc aat cgc cct gct tta aaa 1152 Thr Glu His Asp Ile Ser Ile Phe Ser Ile Asn Arg Pro Ala Leu Lys     370 375 380 caa aaa gaa acg att ctt atg ttc tct gat gaa ccc gat cat aaa gga 1200 Gln Lys Glu Thr Ile Leu Met Phe Ser Asp Glu Pro Asp His Lys Gly 385 390 395 400 gca ctt aaa ccc ggt cat cac cat cac cat cac taa 1236 Ala Leu Lys Pro Gly His His His His His His                 405 410

[0038] <210> 10 <211> 411 <212> PRT <213> Arthrobacter sp. TE1826 <400> 10 Met Ser Ile Lys Lys Asp Tyr Asp Val Ile Val Val Gly Ala Gly Ser   1 5 10 15 Met Gly Met Ala Ala Gly Tyr Tyr Leu Ser Lys Gln Gly Val Lys Thr              20 25 30 Leu Leu Val Asp Ser Phe Asp Pro Pro His Thr Asn Gly Ser His His          35 40 45 Gly Asp Thr Arg Ile Ile Arg His Ala Tyr Gly Glu Gly Arg Glu Tyr      50 55 60 Val Pro Phe Ala Leu Arg Ala Gln Glu Leu Trp Tyr Glu Leu Glu Lys  65 70 75 80 Glu Thr His His Lys Ile Phe Thr Lys Thr Gly Val Leu Val Phe Gly                  85 90 95 Pro Lys Gly Glu Ala Pro Phe Val Ala Glu Thr Met Glu Ala Ala Lys             100 105 110 Glu His Ser Leu Asp Val Asp Leu Leu Glu Gly Ser Glu Ile Asn Lys         115 120 125 Arg Trp Pro Gly Val Thr Val Pro Glu Asn Tyr Asn Ala Ile Phe Glu     130 135 140 Lys Asn Ser Gly Val Leu Phe Ser Glu Asn Cys Ile Arg Ala Tyr Arg 145 150 155 160 Glu Leu Ala Glu Ala Asn Gly Ala Lys Val Leu Thr Tyr Thr Pro Val                 165 170 175 Glu Asp Phe Glu Ile Ala Glu Asp Phe Val Lys Ile Gln Thr Ala Tyr             180 185 190 Gly Ser Phe Thr Ala Ser Lys Leu Ile Val Ser Met Gly Ala Trp Asn         195 200 205 Ser Lys Leu Leu Ser Lys Leu Asn Ile Glu Ile Pro Leu Gln Pro Tyr     210 215 220 Arg Gln Val Val Gly Phe Phe Glu Cys Asp Glu Lys Lys Tyr Ser Asn 225 230 235 240 Thr His Gly Tyr Pro Ala Phe Met Val Glu Val Pro Thr Gly Ile Tyr                 245 250 255 Tyr Gly Phe Pro Ser Phe Gly Gly Cys Gly Leu Lys Ile Gly Tyr His             260 265 270 Thr Tyr Gly Gln Lys Ile Asp Pro Asp Thr Ile Asn Arg Glu Phe Gly         275 280 285 Ile Tyr Pro Glu Asp Glu Gly Asn Ile Arg Lys Phe Leu Glu Thr Tyr     290 295 300 Met Pro Gly Ala Thr Gly Glu Leu Lys Ser Gly Ala Val Cys Met Tyr 305 310 315 320 Thr Lys Thr Pro Asp Glu His Phe Val Ile Asp Leu His Pro Gln Phe                 325 330 335 Ser Asn Val Ala Ile Ala Ala Gly Phe Ser Gly His Gly Phe Lys Phe             340 345 350 Ser Ser Val Val Gly Glu Thr Leu Ser Gln Leu Ala Val Thr Gly Lys         355 360 365 Thr Glu His Asp Ile Ser Ile Phe Ser Ile Asn Arg Pro Ala Leu Lys     370 375 380 Gln Lys Glu Thr Ile Leu Met Phe Ser Asp Glu Pro Asp His Lys Gly 385 390 395 400 Ala Leu Lys Pro Gly His His His His His His                 405 410

[Brief description of drawings]

FIG. 1 is a view showing the construction of a gene encoding a fusion protein.

FIG. 2 is a diagram showing the results of SDS-polyacrylamide gel in which the fusion protein and its selective cleavage state were analyzed.

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

FIG. 4 is a view showing a result of SDS-polyacrylamide gel in which a fusion protein and its selective cleavage state are analyzed.

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[Procedure amendment]

[Submission date] August 8, 2001 (2001.8.8)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Claims

[Correction method] Change

[Correction content]

[Claims]

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0001

[Correction method] Change

[Correction content]

[0001]

TECHNICAL FIELD The present invention relates to a selective cleavage method for obtaining an amine oxidase from a fusion protein. The present invention also relates to a method for obtaining an amine oxidase from a fusion protein, which is characterized by enzymatically cleaving the amino acid sequence of the connecting portion and its peripheral portion. The present invention is
It is useful for a fusion protein which is difficult to be selectively cleaved by a usual method. Further, according to the present invention, it becomes possible to prepare an amine oxidase from a fusion protein easily at a very low cost.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0004

[Correction method] Change

[Correction content]

[0004]

We have found that it is possible to cleave a fusion protein in which a tag and an amine oxidase are linked by using a non-specific endoprotease, and have arrived at the present invention. . More specifically, the inventors have found that by using a non-specific endoprotease, it is possible to obtain an amine oxidase from a fusion protein having a specific amino acid sequence in the connecting portion, and have arrived at the present invention. It has been thought that nonspecific endoprotease does not recognize a specific sequence and cleaves amine oxidase at multiple sites, so it is considered impossible to use it in a method of selectively cleaving a fusion protein. It was However, we conducted various studies and made it possible to obtain an amine oxidase from a fusion protein in which the amino acid sequence of the connecting portion has a specific amino acid sequence using a non-specific endoprotease.

[Procedure amendment 4]

[Document name to be amended] Statement

[Name of item to be corrected] 0005

[Correction method] Change

[Correction content]

That is, the present invention has the following configuration. [1] A method of cleaving a fusion protein in which a tag and an amine oxidase are linked with a non-specific endoprotease. [2] In the step of obtaining the desired amine oxidase by cleaving the sequence of the connecting part or its peripheral part without cutting the sequence of the protein part in the fusion protein in which the tag and the amine oxidase are connected, A method for obtaining an amine oxidase from a fusion protein, which comprises using a specific endoprotease. [3] The method according to [2], wherein the amino acid sequence of the connecting portion is represented by formula (1). X-Met-Phe-Ser- (Xaa) nY (1) [In the formula, X means an amine oxidase and Y means a tag. Xaa means any amino acid residue. n is 0 to 50
Is an integer. [4] The method according to [2], wherein the amino acid sequence of the connecting portion is represented by formula (2). X-Met-Phe-Ser-Asp-Glu-Pro-Asp-His-Lys-Gly-Ala-Leu-Lys- (Xaa) nY (2) [In the formula, X means an amine oxidase and Y is a tag. Means Xaa means any amino acid residue. n is 0-40
Is an integer. [5] The method according to [2], wherein the amino acid sequence of the connecting portion is represented by formula (3). X-Met-Phe-Ser-Asp-Glu-Pro-Asp-His-Lys-Gly-Ala-Leu-Lys-Y (3) [In the formula, X means an amine oxidase and Y means a tag. . [6] The method according to [1] to [5], wherein the tag is an affinity peptide containing adjacent histidine residues. [7] The method according to [1] to [6], wherein the non-specific endoprotease is selected from proteinase K, trypsin, chymotrypsin, and V8 protease. [8] Proteinase K is a non-specific endoprotease
The method according to [7], wherein

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0009

[Correction method] Change

[Correction content]

The property of the amine oxidase to be used in the method of the present invention is not particularly limited, but a single protein having a specific molecular weight is preferable.

[Procedure correction 6]

[Document name to be amended] Statement

[Name of item to be corrected] 0024

[Correction method] Change

[Correction content]

[0024]

EXAMPLES The effects of the present invention will be further clarified by exemplifying the examples of the present invention, but the present invention is not limited thereto. Reference Example 1 Preparation of Gene Encoding Fusion Protein As a desired protein, Bacillus sp. TB, which is a practical enzyme used in a clinical test drug for measuring uric acid
Uricase from 90 strains (urate oxidase, EC 1.7.3.3) was taken up. As a peptide having a specific amino acid sequence, an affinity peptide containing adjacent histidine residues, specifically 6xHis-t in which 6 histidine residues are arranged.
Taking ag, a gene encoding a fusion protein in which this was bound to the carboxyl terminus of uricase via a linking portion was prepared by the following procedure. The flow of creation is also shown in FIG. Plasmid pUOD containing uricase gene (publication of JP-A-2-53488) (Journ
al of Biochemistry, Vol.119, 80-84 (1996)) (Bioindustry, Vol.13, 20-28 (1996)) as a template, and polymerase chain reaction (PCR) under the following composition and conditions. The uricase gene for preparing the fusion protein gene was prepared. DNA polymerase (KOD-Plus- (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 x10 buffer (Toyobo) 5 μl 2 mM dNTP (Toyobo) 5 μl 25 mM MgSO4 (Toyobo) 4 μl Total solution 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 was described in SEQ ID NO: 3 in the sequence listing, and the amino acid sequence of uricase was described in SEQ ID NO: 4 in the sequence listing. The obtained uricase gene is a PCR product purification kit (MagExtractor-PCR
& Gel Clean Up- (manufactured by Toyobo) according to the protocol, and cut with restriction enzymes EcoRI and PstI.
And the expression vector pKK223-3 (manufactured by Amersham Pharmacia) cleaved with PstI and T4 DNA ligase were used for ligation. The resulting uricase gene expression plasmid was named pUODSP1 (FIG. 1). pUO
DSP1 was cleaved with restriction enzymes SmaI and PstI to cleave the carboxyl terminal side of the uricase gene. Synthetic oligonucleotides having the sequences of SEQ ID NOs: 5 and 6 in the sequence listing were ligated to this using T4 DNA ligase, and finally 6xHis-tag in which 6 histidine residues were arranged had a ligation portion at the carboxyl terminal of uricase. An expression plasmid, pUODSP1-HT, containing a gene encoding the fusion protein bound via the plasmid could be constructed (FIG. 1). The sequence of the gene encoding the prepared fusion protein is described in SEQ ID NO: 7 in the sequence listing, and the amino acid sequence of the fusion protein is described in SEQ ID NO: 8 in the sequence listing.

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Reference Example 2 Preparation of Fusion Protein A competent cell of Escherichia coli JM109 (Competent High (manufactured by Toyobo)) was transformed with pUODSP1-HT to prepare a fusion protein producing recombinant bacterium JM109 / pUODSP.
1-HT was prepared. 50 ml of this, TB-brot
Inoculate into a 500 ml volumetric flask containing h and incubate at 37 ° C for 1
Cultured for 7 hours. The bacterial cells were collected from the culture solution by centrifugation, and buffer A (20 mM Tris-HCl (pH 7.
5), after suspending with 350 mM NaCl), the protein was extracted by ultrasonication. Crush the supernatant with nickel chelate
Pass through column chromatography (HiTrap-Chelating- (manufactured by Amersham Pharmacia)) and follow buffer B (20 mM Tris-H) according to the manufacturer's protocol.
Cl (pH 7.5), 350 mM NaCl, 500 m
(M imidazole). Furthermore, the eluate is Se
It is passed through a phasex G-25 desalting / column chromatography (Hitrap-Desalting- (manufactured by Amersham Pharmacia)), eluted with buffer C (20 mM Tris-HCl (pH 7.5)) according to the manufacturer's protocol, and the buffer is replaced. , A purified preparation of the fusion protein was prepared. The analysis results of the disrupted supernatant and the purified protein by SDS-polyacrylamide gel are shown in FIG.

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Reference Example 3 Treatment with Non-Specific Protease and Preparation of Desired Protein A purified preparation of the fusion protein was treated with proteinase K (Nacalai Tesque) 20 mM Tris-HCl (pH 7.
5) Treated with solution. 50 μl fusion protein: about 2 mg
/ Μl proteinase K: about 0.1 to 1 μg / m
It was added at a final concentration of 1 and treated at 37 ° C. for 30 minutes. The reaction was terminated by adding PMSF (phenylmethylsulfonyl fluoride (manufactured by Nacalai Tesque)), which is an inhibitor of proteinase K, at 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, which was the desired protein, could be obtained. The analysis results of the proteinase K-treated product of the fusion protein by SDS-polyacrylamide gel are shown in FIG. The connecting portion of the fusion protein is cleaved by the enzymatic degradation of proteinase K to give 6xH.
The analysis results revealed that the uricase was being prepared by removing the is-tag peptide. Cleavage of the junction of the fusion protein by a non-specific protease
It was also confirmed by mass spectrometry by LDI-TOFF-MASS. It is speculated that the selective cleavage of the fusion protein by a non-specific protease is due to the structural characteristics of the ligation part or the ligation part and its peripheral part, but at this time, the detailed basic mechanism has not been clarified, and its application. Only its utility in terms of aspects is known. Therefore, X-Met-Phe-Ser- (Xaa) nY (1) is characterized in that the amino acid sequence of the linking portion is represented by formula (1) [wherein X represents a protein of 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. Alternatively, 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) [In the formula, X means a protein having a specific molecular weight, and Y means a peptide having a specific amino acid sequence. Xaa means any amino acid residue. n is an integer of 0-40. Alternatively, 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) [In the formula, X means a protein having a specific molecular weight, and Y means a peptide having a specific amino acid sequence. ] The information as a new method was illustrated.

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Reference Example 4 Affinity of Fusion Protein
Preparation of desired protein by binding to beads and treatment with non-specific protease By using the method of this patent, it is possible to bind a fusion protein to affinity beads, which has been substantially difficult in terms of cost, by sequence selective cleavage. This makes it possible to obtain a desired protein. By enabling this technique, high-throughput purification of proteins can be easily carried out. An example of the flow of high throughput protein purification by the method of this patent is shown in FIG. Similar to Reference 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 bacterial cells are collected from the culture solution by centrifugation to collect the buffer A.
After suspending in, the protein was extracted by ultrasonication. Disintegrate liquid with nickel chelate / magnetic beads (MagExtractor-His
-tag- (manufactured by Toyobo)), and the beads were washed with the attached buffer according to the manufacturer's protocol. By this operation, magnetic beads to which the fusion protein is adsorbed are prepared. The magnetic beads were used as proteinase K (manufactured by Nacalai Tesque, Inc.) 20 mM Tris-HCl (pH 7.
5) Treatment with the solution 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, and the mixture was treated 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, which was the desired protein, could be obtained. Proteinase K for magnetic beads
The analysis result of the treated product by SDS-polyacrylamide gel is shown in FIG. The connecting portion of the fusion protein is cleaved by the enzymatic decomposition action of proteinase K to give 6xHi.
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 fusion protein junction by a non-specific protease was also confirmed by mass spectrometry by MALDI-TOFF-MASS. Example 1 Preparation of desired protein by binding fusion protein to affinity bead and treatment with non-specific protease By using the method of this patent, high-throughput purification of amine oxidase can be easily carried out. The sequence of the gene encoding the amine oxidase fusion protein is shown in SEQ ID NO: 9 in the sequence listing, and the amino acid sequence of the fusion protein is shown in SEQ ID NO: 10 in the sequence listing. In the same manner as in Reference Example 4, the recombinant protein producing recombinant protein of amine oxidase was treated with TB-brot.
The cells were inoculated into h and cultured at 37 ° C for 17 hours. The cells were collected from the culture solution by centrifugation, suspended in buffer A, and then sonicated to extract the protein. The disrupted solution was adsorbed on nickel chelate / magnetic beads (MagExtractor-His-tag- (Toyobo)), and the beads were washed with the attached buffer according to the manufacturer's protocol. By this operation, magnetic beads to which the fusion protein is adsorbed are prepared. This magnetic bead was used as Proteinase K (manufactured by Nacalai Tesque, Inc.)
Treatment with a mM Tris-HCl (pH 7.5) solution eluted the desired amine oxidase. Magnetic beads 2
About 1 to 4 μg of proteinase K solution for 0 μl volume
100 μl was added at a concentration of / ml and treated 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,
The desired amine oxidase could be obtained. Since magnetic beads are used in this example, solid-liquid separation can be performed very easily by using a magnet without performing centrifugation. The solid-liquid separation system for magnetic beads using a magnet is easy to automate, and in fact, an automation device has already been developed and sold (MFX-2000, MFX-6000, MF
X-9600 (made by Toyobo)). Therefore, by combining these apparatuses with the method of the present patent, high-throughput purification of proteins can be easily realized.

Claims (9)

[Claims] 1. A fusion protein in which a tag and a protein are linked,
A method of cleaving with a non-specific endoprotease. 2. A non-specific method for a fusion protein in which a tag and a protein are ligated, in a step of obtaining a desired protein by cleaving the sequence of the ligation part or its peripheral part without cleaving the sequence of the protein part. 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 sequence easily recognized by the non-specific endoprotease is represented by the formula (1). X-Met-Phe-Ser- (Xaa) nY (1) [In the formula, X means a protein and Y means 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 sequence easily recognized by the non-specific endoprotease is represented by the formula (2). X-Met-Phe-Ser-Asp-Glu-Pro-Asp-His-Lys-Gly-Ala-Leu-Lys- (Xaa) nY (2) [where X is a protein and Y is a tag] To do. Xaa means any amino acid residue. n is an integer of 0-40. ] 5. The method according to claim 2, wherein the sequence that is easily recognized by the non-specific endoprotease is represented by the formula (3). X-Met-Phe-Ser-Asp-Glu-Pro-Asp-His-Lys-Gly-Ala-Leu-Lys-Y (3) [wherein X represents a protein and Y represents a tag. ] 6. The method according to claim 1, wherein the tag is an affinity peptide containing adjacent histidine residues. 7. The method according to claim 1, wherein the protein is an amine oxidase. 8. The method according to claim 1, wherein the non-specific endoprotease is selected from proteinase K, trypsin, chymotrypsin and V8 protease. 9. The method according to claim 8, wherein the non-specific endoprotease is proteinase K.