JP3145431B2 - Method for producing stable isotope-labeled protein and reagent kit - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a stable isotope-labeled protein used in the fields of molecular biology and protein engineering, and more particularly, to labeling a protein with a stable isotope, In a technique for analyzing the three-dimensional structure of a protein or its function using a resonance device or the like, a method of labeling only a specific type of amino acid residue or all amino acid residues of a protein, and a reagent for performing the method About the kit.

[0002]

2. Description of the Related Art Conventionally, in the fields of molecular biology and protein engineering, carbon 13 (hereinafter referred to as ¹³ C), nitrogen 15
(Hereinafter ¹⁵ referred to as N) labeled protein with a stable isotope, such as a technique for analyzing the three-dimensional structure and its function of the protein is known with the labeled protein. In addition, several examples of a method for producing a protein labeled with such a stable isotope are known.

[0003] When cells such as bacteria and animal cells that synthesize and secrete a target protein are available, the cells are cultured in an amino acid mixed medium containing a specific stable isotope-labeled amino acid, and the synthesized target protein is synthesized. Separates and produces proteins.

When the base sequence encoding the genetic information of the target protein is known, the base sequence is incorporated into a vector by gene recombination, and the vector is introduced into a host to express the target protein. Then, the target protein is separated and purified. At this time, by using a mutant requiring the amino acid to be labeled as a host bacterium and adding the stable isotope-labeled amino acid to the medium,
A protein in which the target amino acid residue is labeled with a stable isotope can be obtained.

When all amino acid residues are labeled, the carbon source, nitrogen source, oxygen source, hydrogen source, sulfur source, etc. in the medium are replaced with those labeled with stable isotopes to synthesize the target protein. The target cell or the cell transformed to synthesize the target protein is cultured to synthesize the target protein, and then the target protein is separated and purified.

[0006]

However, the conventional method for producing a stable isotope-labeled protein has the following problems because the protein is synthesized using an intracellular protein synthesis system.

A. If the cell that synthesizes the protein is not an amino acid-requiring mutant, the cell itself has an amino acid biosynthesis system, and the labeling rate of stable isotopes by amino acids newly synthesized in the cell may be 40% in some cases. To a degree.

B. In addition, in vivo, since a metabolic pathway of amino acids exists, a labeled stable isotope is transferred to an undesired amino acid. Especially ¹⁵ N labeled on the amino group
Is remarkably transferred by aminotransferase. Also, metabolic reactions between serine ← → glycine ← → threonine occur very frequently, and it is difficult to label only specific amino acids among these three amino acids.

C. In addition, when a gene recombination technique is used, as a general problem of gene recombination, it often happens that a target protein cannot be obtained due to insolubilization of the protein or degradation of the protein by intracellular protease.

D. In addition, since a stable isotope-labeled amino acid is added as a medium, the amino acid is consumed not only for the purpose of protein synthesis but also for cell growth. For this reason, a large amount of stable isotope-labeled amino acids is required, so that the target protein obtained is very expensive.

On the other hand, a technique for synthesizing a radioisotope-labeled protein using a cell-free protein synthesis system obtained by extracting a protein synthesis system from the inside of a cell is already known, and a reagent kit therefor is also commercially available. However, since the cell-free protein synthesis system used in this reaction uses a conventional batch-type reaction system, only a trace amount of protein (μg or less) can be synthesized, and a system using a radioactive isotope can be used. Otherwise, it cannot be detected. For this reason, in the prior art, it has not been considered at all to synthesize a stable isotope-labeled protein with high safety in place of a radioactive isotope in a cell-free protein synthesis system. Reagent kits that are already commercially available also aim only to synthesize radioisotope-labeled proteins in a batch-type cell-free protein synthesis system, and synthesize stable isotope-labeled proteins in a continuous cell-free protein synthesis system. Not suitable for purpose.

The present invention has been made in view of the above circumstances, and in the synthesis of a stable isotope-labeled protein, 20 kinds of amino acid residues present in a protein are not insolubilized or degraded by an intracellular protease. Nearly 100 at any atom of any particular amino acid residue
It is an object of the present invention to provide a method for producing a stable isotope-labeled protein in which a target protein labeled with a stable isotope at a% labeling rate can be obtained in an inexpensive, simple and necessary and sufficient amount.

[0013]

Means for Solving the Problems The present invention is intended to continuously supply a substrate solution containing an amino acid to a cell-free protein synthesis system, and take out an effluent containing a protein produced from the reaction system to obtain a continuous solution. A method for producing a protein, comprising:
The problem has been solved by a method for producing a stable isotope-labeled protein, which comprises using a substrate containing at least one kind of labeled amino acid labeled with a stable isotope such as ¹³ C, ¹⁵ N.

Further, the method for producing a stable isotope-labeled protein includes a substrate containing at least one kind of labeled amino acid labeled with a stable isotope such as ¹³ C, ¹⁵ N, a cell-free protein synthesis system, an ATP, The present invention can be applied to a reagent kit for a stable isotope-labeled protein including ribonucleotides such as GTP, CTP, and UTP.

The cell-free protein synthesis system is preferably a cell-free protein synthesis system having reduced activity other than the protein synthesis system such as an amino acid biosynthesis system and an amino acid metabolism system. Cell-free protein synthesis systems with reduced activities other than this protein synthesis system include cell-free protein synthesis systems extracted from bacteria or cells cultured under amino acid-rich conditions, inhibitors of amino acid biosynthesis systems and amino acid metabolism systems. Cell-free protein synthesis system to which amino acids are added, cell-free protein synthesis system extracted from bacteria or cells deficient in amino acid biosynthesis system and amino acid metabolism system, cell-free system except for amino acid biosynthesis system and amino acid metabolism system by monoclonal antibody One or a combination of two or more of various cell-free protein synthesis systems of the protein synthesis system can be used.

[0016]

A method for producing a stable isotope-labeled protein by continuously supplying a substrate containing at least one type of labeled amino acid labeled with a stable isotope such as ¹³ C or ¹⁵ N using a cell-free protein synthesis system. As a result, a target protein in which a stable isotope is labeled at a labeling rate of almost 100% only at an arbitrary atom of an arbitrary specific amino acid residue among 20 kinds of amino acid residues present in a protein can be obtained at low cost.

[0017]

Examples The cell-free protein synthesis system preferably used in the present invention includes a cell-free translation system such as a rabbit reticulocyte hemolysis system, a wheat germ extract, and a cell-free transcription system such as an E. coli S-30 extract. Such as a translation conjugate system. These are mainly ribosomes, enzymes required for tRNA and protein synthesis, and synthesize proteins using 20 types of amino acids as substrates.

Further, in the present invention, those having reduced activities other than the protein synthesis system such as the amino acid biosynthesis system and the amino acid metabolism system existing in the cell-free protein synthesis system are used. As a method for preparing a cell-free protein synthesis system with reduced activity other than the protein synthesis system, cells or bacteria from which the cell-free protein synthesis system is to be removed are subjected to amino acid-rich conditions (under normal amino acid concentration in culture medium). A medium having a concentration about 10 times higher is used) to reduce the activity of the cells or the body of the bacterium other than the protein synthesis system such as the amino acid biosynthesis system and the amino acid metabolism system. (1973) Annu. Rev. G
enet. 7, 267-287) and a method known in the art, such as a cell-free transcription / translation conjugation system or a method for preparing tRNA, which is separated from bacteria or cells and used.

Other methods for preparing a cell-free protein synthesis system with reduced activity other than the protein synthesis system include the following methods:・ Using a cell-free protein synthesis system extracted from bacteria or cells deficient in amino acid biosynthesis or amino acid metabolism, etc. ・ Preparing antibodies (monoclonal antibodies) against amino acid biosynthesis or amino acid metabolism There is a method using a cell-free protein synthesis system excluding amino acid biosynthesis system and amino acid metabolism system using the antibody. These methods are used alone or in combination of two or more to reduce the activity other than the protein synthesis system. Obtain a cellular protein synthesis system.

The substrate used in the present invention includes 20 kinds of amino acids constituting a protein (glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, glutamic acid, asparagine, glutamine, lysine, arginine, A substrate solution containing a labeled amino acid in which at least one or all of amino acids of cystine, methionine, phenylalanine, tyrosine, tryptophan, histidine, and proline) are labeled with a stable isotope such as ¹³ C or ¹⁵ N is used. Can be This substrate solution contains a labeled amino acid,
In addition to unlabeled amino acids, ATP, GTP, CTP, UTP
And the like.

FIG. 1 is a view showing an example of a production apparatus suitable for carrying out the method for producing a stable isotope-labeled protein according to the present invention, wherein reference numeral 1 denotes a reaction tank, 2 denotes a substrate solution tank, and 3 denotes a substrate solution tank. Is the effluent. The reaction tank 1 contains a cell-free protein synthesis system having reduced enzyme activity other than the above-described protein synthesis system. The substrate solution pumped from the substrate solution tank 2 is continuously supplied from the lower side of the reaction tank 1, and the label generated in the reaction tank 1 is passed through the ultrafilter 4 attached to the upper part of the reaction tank 1. Effluent 3 containing protein
Is spilled.

In order to continuously produce a labeled protein using this apparatus, a solution containing a cell-free protein synthesis system for synthesizing a target protein is accommodated in the reaction tank 1. The substrate solution containing the labeled amino acid is continuously pumped. In order to pump the substrate solution into the reaction tank 1, a pump that does not contain a gas phase in the system, has a small pulsating flow, and can control the pressure and the flow rate, for example, a pump for high-performance liquid chromatography, a medium-pressure liquid It is desirable to pump using a pump for chromatography, a pump for low pressure liquid chromatography, or the like. The pumping amount of the substrate solution is usually set to be constant, but the pumping amount may be increased or decreased as the reaction time elapses.

The 20 kinds of amino acids containing at least one kind of labeled amino acid supplied into the reaction vessel 1 are biosynthesized into a labeled protein containing the labeled amino acid by the action of a cell-free protein synthesis system. In this labeled protein, at least one of the amino acids constituting the protein is labeled with a stable isotope such as ¹³ C or ¹⁵ N, and the labeled amino acid residue can be detected by using NMR.

The effluent 3 containing the labeled protein biosynthesized in the reaction tank 1 passes through the ultrafilter 4 and is collected in the collection container 5. The ultrafiltration device 3 does not allow the permeation of the cell-free protein synthesis system itself, such as ribosomes, RNA, enzymes, etc., contained in the reaction tank 1, and synthesizes the synthesized labeled protein, substrate, or degradation products thereof (AMP, GMP). , A polyphosphate, an inorganic phosphate, etc.) are used.

The substrate solution in the substrate solution tank 2 is 10
It is desirable to store at a temperature of not more than ℃, and it is desirable to keep the temperature in the reaction vessel 1 at 20 to 40 ℃. Further, the inside of the reaction tank 1 may be stirred by using a magnetic stirrer or the like.

As a method for separating and purifying the labeled protein from the effluent 3, a conventionally known method for separating and producing a protein is used, for example, ion exchange chromatography, gel filtration chromatography, affinity chromatography, electrophoresis and the like. Is preferably used. The separated labeled protein is used in technology to detect labeled amino acid residues by nuclear magnetic resonance (NMR) and analyze its steric structure and function, and is also used in medical fields such as metabolic test reagents. Available.

The method for producing the stable isotope-labeled protein is as follows:
By producing a stable isotope-labeled protein by continuously supplying a substrate containing at least one kind of labeled amino acid labeled with a stable isotope such as ¹³ C or ¹⁵ N using a cell-free protein synthesis system, Out of the 20 types of amino acid residues present in the above, a stable labeled isotope can be produced at a labeling rate of almost 100% at any given atom of any given amino acid residue.

Next, a reagent kit for a stable isotope-labeled protein according to the present invention will be described. This reagent kit includes an amino acid mixture (amino acid substrate) containing at least one kind of labeled amino acid labeled with a stable isotope such as ¹³ C or ¹⁵ N. Cell-free protein synthesis system ATP, GTP, CTP, UTP, or other ribonucleotides And is provided. In addition to these materials, a buffer solution for adjusting the pH of the substrate solution and the like can be included.

In the analysis of protein structure, etc., many types of labeled proteins having different types of labeled amino acids are often required. In such a case, it is desirable to prepare an amino acid mixture for a substrate in which various kinds of labeled amino acids are combined. That is, it is an amino acid mixture using a labeled amino acid for at least one of the 20 amino acids, or an amino acid mixture using a labeled amino acid for all of the 20 amino acids. Although various combinations of amino acid mixtures containing one or more labeled amino acids are conceivable, usually one of the 20 amino acids is a labeled amino acid and the remaining 19 are unlabeled (natural). Combinations of the following combinations are used: an amino acid mixture of amino acids (20 types), an amino acid mixture of all types of amino acids as labeled amino acids, and an amino acid mixture of all types of amino acids as unlabeled (natural) amino acids. .

As the above-mentioned cell-free protein synthesis system, it is desirable to use a cell-free protein synthesis system with reduced activity other than the protein synthesis system. Such a cell-free protein synthesis system includes a cell-free protein synthesis system extracted from bacteria or cells cultured under amino acid-rich conditions, a cell-free protein synthesis system added with an inhibitor for an amino acid biosynthesis system or an amino acid metabolism system,
Cell-free protein synthesis system extracted from bacteria or cells deficient in amino acid biosynthesis or amino acid metabolism, or cell-free protein synthesis in which amino acid biosynthesis or amino acid metabolism is excluded by monoclonal antibodies One or more of the synthetic systems can be used in combination.

In this reagent kit, the amino acid mixture, the cell-free protein synthesis system, and the ribonucleotide
Each of them is housed in a separate container, or the substrate in which the amino acid mixture and the ribonucleotide are mixed and the cell-free protein synthesis system are housed in separate containers. The amino acid mixture and the ribonucleotide are relatively stable when stored in a frozen state even in a state of being dissolved in water (buffer solution or acid solution).
The cell-free protein synthesis system is a liquid containing ribosomes, RNAs, enzymes, and the like extracted from bacteria and cells, and degrades in a short time as it is.
It is desirable to store the solution in liquid nitrogen.

(Experimental Example) The manufacturing apparatus shown in FIG.
The synthesis of the labeled protein was performed. As a cell-free protein synthesis system, a transcription / translation coupling system using an S30 extract of Escherichia coli developed by Zubay et al. (Zubay G. (1973))
Annu. Rev .. Genet. 7, 267-287)
The reference was synthesized CAT labeled with ¹⁵ N glycine residues (chloramphenicol acetyl transferase).

Preparation of Escherichia coli S30 Extract A Escherichia coli A19 strain was prepared according to the method of Zubay et al. Note that this extract could be stored for a relatively long time if it was at -80 ° C or lower.

In the plasmid DNA transcription / translation conjugation system, a plasmid DNA pACL6 prepared to efficiently express CAT was used.

The continuous cell-free protein synthesis reaction has a capacity of 1 m.
1 reaction vessel. Here, 170 μl of E. coli S3
0 extract, 100 μg of plasmid DNA, 174 μg
Was filled, and the substrate solution was filled. This reaction vessel was heated to 37 ° C. and subjected to high performance liquid chromatography (HPL).
The substrate solution was supplied using a pump for C) (manufactured by Tosoh Corporation, CCPM).

As the substrate solution, 20 kinds of amino acids each containing glycine labeled with ¹⁵ N (0.35 mmol each), a 55.0 mmol tris acetic acid solution (pH 8.2), 1.65 mmol・ DTT, 1.2
2 mmol ATP, 0.84 mmol ・ CTP ・ GTP
-UTP, 27.0 mmol-phosphoenolpyruvate, 1.9% polyethylene glycol-6000,
34.4 μg / ml folinic acid, 0.64 mmol
A solution containing 3 ′, 5′-cyclic AMP, 36.0 mmol ammonium acetate, 72.0 mmol potassium acetate, 9.7 mmol calcium acetate, 10.0 mmol magnesium acetate was used. This substrate solution was able to be stored for a long period of time by freezing at -20 ° C.

At the same time as supplying the substrate solution, the reaction product (labeled CAT) and the ribonucleotides, amino acids and degradation products (labeled CAT) used in the reaction were passed through an ultrafiltration membrane YM100 (manufactured by Amicon) having a molecular weight cut off of 100,000 daltons. An effluent containing AMP, GMP, inorganic phosphoric acid, etc.) was taken out of the reactor. The supply amount of the substrate solution was 2 ml.
/ Hour. The substrate solution was stored at about 4 ° C.

Using such a system, a CAT (stable isotope-labeled protein) in which glycine residues of 20 kinds of amino acids were labeled with ¹⁵ N was synthesized for 17 hours by a cell-free protein synthesis system. . After completion of the synthesis, CAT in the effluent was purified by affinity chromatography.

[0039] 24 hours CAT after purification in 6N-HCl, and acid hydrolysis, and the obtained amino acid was derivatized with TBDMS, result of analysis of each amino acid in GC-MS, only glycine residues ¹⁵ N It was confirmed that it was labeled with.

[0040]

As described above, the present invention uses a cell-free protein synthesis system to continuously provide a substrate containing at least one type of labeled amino acid labeled with a stable isotope such as ¹³ C or ¹⁵ N. Providing a stable isotope-labeled protein by supplying the same enables stable isotope labeling with almost 100% efficiency on any atom of any specific amino acid residue among 20 types of amino acid residues present in the protein. The target labeled protein thus produced can be produced at low cost. Therefore, it is possible to safely and extremely easily produce a labeled protein used for a technique for analyzing the three-dimensional structure and function of a protein, and this greatly contributes to a technique for analyzing a protein structure and the like.

[Brief description of the drawings]

FIG. 1 is a schematic configuration diagram of a production apparatus for explaining a method for producing a stable isotope-labeled protein of the present invention.

[Explanation of symbols]

 Reference Signs List 1 reaction tank 2 substrate solution tank 3 effluent 4 ultrafilter

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-62-130692 (JP, A) JP-A-1-503119 (JP, A) JP-A-3-503479 (JP, A) International publication 91/2075 (WO, A1) International Publication No. 91/2076 (WO, A1) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12P 21/00-21/02 BIOSIS (DIALOG) WPI (DIALOG)

Claims (12)

(57) [Claims] 1. An amino acid biosynthesis system, an amino acid metabolism system, etc.
While continuously supplying an amino acid-containing substrate solution to a cell-free protein synthesis system having reduced enzyme activity other than the protein synthesis system of the above, the effluent containing the protein produced from the reaction system is taken out and the target protein is continuously obtained. A method for producing a stable isotope-labeled protein, wherein the substrate comprises at least one kind of labeled amino acid labeled with a stable isotope such as ¹³ C or ¹⁵ N. 2. The method according to claim 2, wherein the cell-free protein synthesis system comprises an amino acid.
Extracted from bacteria or cells cultured under acid-rich conditions
The method for producing a stable isotope-labeled protein according to claim 1, wherein the cell-free protein synthesis system is used. 3. The method according to claim 1, wherein the cell-free protein synthesis system comprises an amino acid.
Claim acid biosynthesis system, the use of the added <br/> cell-free protein synthesis system inhibitors to amino acid metabolism characterized 1
A method for producing the stable isotope-labeled protein according to the above. 4. A bacterium deficient in an amino acid biosynthesis system, an amino acid metabolism system, or the like as the cell-free protein synthesis system.
The method for producing a stable isotope-labeled protein according to claim 1 , wherein a cell-free protein synthesis system extracted from cells is used. As claimed in claim 5 wherein said cell-free protein synthesis system, monochromator
Amino acid biosynthesis system, amino acid metabolism system by lonal antibody
2. The method for producing a stable isotope-labeled protein according to claim 1 , wherein a cell-free protein synthesis system excluding the above is used. 6. The cell-free protein synthesis system according to claim 2
Use in combination of at least two of the above
The method for producing a stable isotope-labeled protein according to claim 1, characterized in that: 7. Labeling with a stable isotope such as ¹³ C and ¹⁵ N
A substrate containing at least one type of labeled amino acid,
Protein synthesis systems such as amino acid biosynthesis and amino acid metabolism
Cell-free protein synthesis system with reduced activity other than
With ribonucleotides such as P, GTP, CTP, and UTP
A reagent kit for a stable isotope-labeled protein, comprising: 8. The cell-free protein synthesis system according to claim 8, wherein
Extracted from bacteria or cells cultured under acid-rich conditions
Claim to feature the use of cell-free protein synthesis system was
A reagent kit for a stable isotope-labeled protein according to claim 7 . As claimed in claim 9 wherein said cell-free protein synthesis system, according to claim 7 which comprises using amino acid biosynthetic, the added <br/> cell-free protein synthesis system inhibitors to amino acid metabolism
A reagent kit for the stable isotope-labeled protein according to the above. As claimed in claim 10 wherein said cell-free protein synthesis system, Ami
The reagent kit for a stable isotope-labeled protein according to claim 7 , wherein a cell-free protein synthesis system extracted from a bacterium or a cell deficient in a no acid biosynthesis system, an amino acid metabolism system, or the like is used. As claimed in claim 11 wherein said cell-free protein synthesis system, mono-
Amino acid biosynthesis, amino acid metabolism by clonal antibody
The reagent kit for a stable isotope-labeled protein according to claim 7 , wherein a cell-free protein synthesis system excluding a system and the like is used. 12. The cell-free protein combination according to claims 8 to 11.
The reagent kit for a stable isotope-labeled protein according to claim 7 , wherein at least two or more of the adult systems are used in combination .