JPWO2008096846A1 - Composition for stable isotope-labeled protein synthesis and method for producing stable isotope-labeled protein - Google Patents

Abstract

An object of the present invention is to provide a means for inexpensively synthesizing a stable isotope-labeled protein useful in the field of nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry. According to the present invention, (A) L-alanine, L-arginine, L-aspartic acid, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L- A mixture of 16 amino acids: phenylalanine, L-proline, L-serine, L-threonine, L-tyrosine, L-valine, (B) indole, (C) hydrogen sulfide or sodium sulfide, and (D) ammonium A salt, and / or at least one of the amino acids in the amino acid mixture and / or any or all of indole, hydrogen sulfide or sulfide salt, ammonium salt is labeled with a stable isotope The present invention provides a composition for synthesizing stable isotope-labeled proteins, and a method for synthesizing stable isotope-labeled proteins using the composition.

Description

  The present invention relates to a composition for synthesizing a stable isotope-labeled protein useful in the field of nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry, and a method for producing a stable isotope-labeled protein using the composition.

Protein samples used for protein structure and function analysis are labeled with stable isotopes. For example, when conducting a three-dimensional structure analysis of a protein in nuclear magnetic resonance spectroscopy (NMR) analysis, a stable isotope such as ¹³ C, ¹⁵ N, ² H, or ¹⁷ O is introduced into the target protein, thereby increasing the measurement sensitivity. The effect of improving analysis and facilitating analysis can be obtained. In addition, when a protein is quantitatively or qualitatively analyzed in mass spectrometry, a stable isotope ( ¹³ C, ¹⁵ N, ² H, ¹⁸ O, etc.) labeled protein is used as an internal standard.

Methods for introducing stable isotopes into proteins have been studied for a long time, and several methods have been implemented in the above-described analytical field. The most common method, labeled carbon source with a stable isotope of the recombinant E. coli (e.g., glucose - ¹³ C) or nitrogen source (e.g. ammonium chloride - ¹⁵ N) were cultured in mineral medium containing, in a known manner Examples include a method in which a large amount of recombinant protein is expressed in cells, the recovered E. coli is disrupted and extracted by an appropriate method, and a desired stable isotope-labeled protein is purified from this extract. A stable isotope-labeled protein synthesis method using live cells such as yeast, animal cells, and insect cells instead of Escherichia coli as a host is also widely used.

  Furthermore, in recent years, cell-free protein synthesis technology has been established that separates functional components that synthesize proteins from crushed cell extracts without using live cells and efficiently synthesizes stable isotope-labeled proteins in vitro. (Non-patent Document 1 and Patent Document 1). The cell-free protein synthesis system can efficiently synthesize stable isotope-labeled proteins from stable isotope-labeled amino acids, and there are no problems such as growth inhibition due to the deuterium isotope effect found in living cells. There are advantages such as being suitable for the production of many kinds of proteins.

  When a stable isotope labeled protein is synthesized by such a cell-free protein synthesis system, it is necessary to use 20 kinds of stable isotope labeled amino acids as substrate amino acids. There are several known methods for producing stable isotope-labeled amino acids depending on the type of stable isotope. For example, in the case of carbon / nitrogen stable isotopes, stable isotope-labeled nutrient sources ( Protein in a medium containing glucose, carbon dioxide, acetic acid, methanol, nitrate, ammonium salt, etc.) in the case of deuterium stable isotopes in heavy water and oxygen stable isotopes in microorganisms cultured in oxygen isotope-labeled water. In general, extraction, purification, acid hydrolysis, or enzymatic degradation with protease, exo (endo) peptidase or the like, the amino acid is decomposed and separated and purified using a cation exchange column or the like. However, in the method of degrading proteins with the above enzymes, it is difficult to completely degrade the amino acid unit, and amino acid dimers and trimers become impurities, which inhibit cell growth and cell-free protein synthesis reaction. (Non-Patent Document 2). In addition, the above enzyme has a problem of causing autolysis and lowering the isotope labeling rate of amino acids. On the other hand, in the method of producing by acid hydrolysis, it can be decomposed into amino acids relatively efficiently and isotope dilution does not occur, but the four types of amino acids L-tryptophan, L-cysteine, L-asparagine, and L-glutamine are Since it is extremely unstable to acid, it decomposes and disappears in the acid hydrolysis process. Therefore, the stable isotope-labeled amino acids produced by the acid hydrolysis method are only 16 types of amino acids except the above-mentioned 4 types of amino acids. Therefore, the stable isotope labeling of the four amino acids L-tryptophan, L-cysteine, L-asparagine, and L-glutamine must be separately synthesized by other methods such as fermentation, chemical synthesis, and enzymatic synthesis. Very expensive and expensive.

In particular, oxygen isotope ⁽¹⁷ O) labeled amino acid in which the effect is expected in the solid-state NMR fields in recent years attention, when labeled amino acids with oxygen isotope ⁽¹⁷ O), oxygen isotope ⁽¹⁷ O) labeled Since the reaction is carried out under strong acid conditions in which water is saturated with hydrogen chloride gas, there is a problem that the above four amino acids that are extremely unstable to acids are decomposed. Therefore, although an oxygen isotope ( ¹⁷ O) -labeled amino acid has been established as a production method, a product at an economically practical level is not yet commercially available. As a means for solving this, for example, Fmoc- (or tBoc-) amino acid in which a carboxyl group is derivatized with an ester compound is hydrolyzed in a solvent containing oxygen isotope ( ¹⁷ O) -labeled water to form carboxyl. A technique of labeling one oxygen atom in a group with an oxygen isotope ( ¹⁷ O) has been proposed (Patent Document 2). As a result, oxygen isotope labeling was possible for the above-mentioned four acid-labile amino acids. Furthermore, by repeating the esterification of the carboxyl group and the hydrolysis in the presence of oxygen-labeled water, it is possible to label all two oxygen atoms of the carboxyl group with an oxygen isotope. This amino acid has been converted to Fmoc- (or tBoc-), and is excellent in that it can be chemically polymerized directly using a peptide synthesizer to synthesize an oxygen isotope-labeled protein. However, in order to carry out the above method and label all the oxygen atoms of the carboxyl group with an oxygen isotope, it is necessary to repeat the esterification reaction and hydrolysis of the carboxyl group several times, which requires a lot of labor. Loss due to purification occurs. In addition, in order to synthesize proteins with higher molecular weights and complex higher-order structures, it is complicated in that a step of preparing amino acids by purification after removing Fmoc- (or tBoc-) derivatives is necessary. .

As described above, the stable isotope-labeled protein synthesis method using cell-free protein synthesis technology can efficiently use stable isotope-labeled amino acids, and has achieved certain results from an economic point of view. Since some stable isotope-labeled amino acids including the above-mentioned oxygen isotope-labeled amino acids are expensive, it is still not economically practical.
JP 2000-175695 A JP 2006-8666 Kigawa T. et al., Cell-free production and stable-isotope labeling of milligram quantities of proteins, FEBS Lett., 442 (1), 15-9, 1999 Andrew P. Hansen, et al., A practical method for uniform isotopic labeling of recombinant proteins in mammalian cells, Biochemistry, 31 (51), 12713-12718, 1992

  Accordingly, an object of the present invention is to provide means for synthesizing a stable isotope-labeled protein at a low cost.

  As a result of intensive studies to solve the above problems, the present inventors have found that 16 kinds of amino acids (-alanine, L-arginine, L-aspartic acid, L-glutamic acid, glycine, L-histidine, L-isoleucine, In the reaction system containing L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tyrosine, L-valine), four types of amino acids (L- By adding the precursor substances (tryptophan, L-cysteine, L-asparagine, L-glutamine), the stable isotope labels of the above four types of amino acids, which have been difficult and expensive to manufacture, can be added. At least, it was found that a stable isotope-labeled protein of the same level as when 20 kinds of amino acids were added could be synthesized inexpensively and efficiently, and the present invention was completed.

  That is, the present invention includes the following inventions.

(1) (A) L-alanine, L-arginine, L-aspartic acid, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L -Proline, L-serine, L-threonine, L-tyrosine, L-valine amino acid mixture consisting of 16 kinds of amino acids, (B) indole, (C) hydrogen sulfide or sulfide salt, and (D) ammonium salt A stable isotope, characterized in that at least one of the amino acids in the amino acid mixture and / or at least one of indole, hydrogen sulfide or sulfide salt, ammonium salt is labeled with a stable isotope Composition for body-labeled protein synthesis.

(2) The stable isotope according to (1), wherein the stable isotope is one or a combination of two or more selected from the group consisting of ¹⁷ O, ¹⁸ O, ¹³ C, ¹⁵ N, and ² H Composition for labeling protein synthesis.

(3) A reagent kit for the synthesis of a stable isotope-labeled protein comprising the composition according to (1) or (2).

(4) A method for producing a stable isotope-labeled protein, comprising synthesizing a stable isotope-labeled protein using the composition according to (1) or (2).

(5) The production method according to (4), wherein the synthesis of the stable isotope-labeled protein is performed in a cell-free protein synthesis system.

(6) The production method according to (4), wherein the stable isotope-labeled protein is synthesized in a living cell.

  According to the present invention, the stable isotope labels of four types of amino acids L-tryptophan, L-cysteine, L-asparagine, and L-glutamine, which have conventionally been costly to manufacture, can be used without using these amino acids. It was possible to convert from other amino acids in the protein synthesis reaction system, and to produce by in-system synthesis. Therefore, the present invention is very useful as a technique for synthesizing an inexpensive and efficient stable isotope labeled protein.

This shows the synthesis of CAT protein by a cell-free protein synthesis system using a mixture of 19 amino acids (without L-tryptophan) and indole. This shows the synthesis of CAT protein by a cell-free protein synthesis system using a mixture of 19 amino acids (without L-cysteine) and sodium sulfide. A mixture of 18 amino acids (without L-cysteine and L-tryptophan) or 16 amino acids (without L-asparagine, L-glutamine, L-cysteine and L-tryptophan) and no sodium sulfide and indole The synthesis of CAT protein by the cell protein synthesis system is shown. Shows the ¹⁵ N-HSQC spectrum of Ras protein (top: synthesis with 20 amino acid mixtures, bottom: 16 amino acid mixtures + unlabeled indole, sodium sulfide, sodium acetate- ¹⁵ N) Shows TOF-MS measurement of ¹⁸ O-labeled protein (UBA protein) (A: unlabeled 16 amino acid mixture + indole, sodium sulfide, B: ¹⁸ O labeled 16 amino acid mixture + unlabeled asparagine, glutamine, tryptophan, Cysteine, C: ¹⁸ O-labeled 16 amino acid mixture + indole, sodium sulfide).

  This application claims the priority of the Japan patent application 2007-030650 for which it applied on February 9, 2007, and includes the content described in the specification of this patent application.

  Hereinafter, the present invention will be described in detail.

1. Composition for Stable Isotope Labeled Protein Synthesis According to the present invention, a composition for stable isotope labeled protein synthesis is provided. The composition for synthesizing stable isotope-labeled protein comprises an amino acid mixture comprising 16 kinds of amino acids and an indole, a hydrogen sulfide or sulfide salt, and an ammonium salt, which are precursors of four kinds of amino acids, and the amino acid mixture. It is characterized in that at least one of the amino acids therein and / or at least one of indole, hydrogen sulfide or sulfide salt, and ammonium salt is labeled with a stable isotope.

  The 16 types of amino acids in the above “amino acid mixture” are L-alanine, L-arginine, L-aspartic acid, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L -Methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tyrosine, L-valine. The amino acid mixture may be a mixture of 16 types of amino acids, or a commercially available amino acid mixture.

  Further, the above “four kinds of amino acids” refer to L-tryptophan, L-cysteine, L-asparagine, and L-glutamine. Many microorganisms that do not require essential amino acids have an amino acid biosynthetic system for synthesizing all amino acids constituting a protein in cells.

  As shown below, L-tryptophan is tryptophan synthase (EC4.2.1.20) or tryptophanase (EC 4.1.99.1) from its precursors pyruvate, ammonium ions, and indole, or from L-serine and indole. ). L-cysteine is o-acetylated by the action of serine o-acetyltransferase (EC2.3.1.30) to become o-acetyl L-serine, and L-cysteine is produced by the action of cysteine synthase (EC4.2.99.8). Reacts with hydrogen sulfide to produce L-cysteine.

また、下記に示すように、L-アスパラギン、L-グルタミンは、L-アスパラギン酸、L-グルタミン酸の側鎖の水酸基が、アスパラギンシンセターゼ(E.C.6.3.1.1)及びグルタミンシンセターゼ(E.C.6.3.1.2)の作用によりアミノ基に置換されることで生成される。

Further, as shown below, L-asparagine, L-glutamine, L-aspartic acid, hydroxyl group of the side chain of L-glutamic acid, asparagine synthetase (EC6.3.1.1) and glutamine synthetase (EC6.3.1) It is generated by substitution with an amino group by the action of .2).

従って、上記の「4種類のアミノ酸の前駆物質」とは、具体的には、L-トリプトファンの前駆物質であるインドール、L-システインの前駆物質である硫化水素または硫化物塩、L-アスパラギン、L-グルタミンの前駆物質であるアンモニウム塩をいう。

Therefore, the above-mentioned “four amino acid precursors” specifically include indole, which is a precursor of L-tryptophan, hydrogen sulfide or sulfide salt, which is a precursor of L-cysteine, L-asparagine, An ammonium salt that is a precursor of L-glutamine.

  Here, the ammonium salt is not particularly limited as long as it has an action of becoming a donor of an amino group in a metabolic system from aspartic acid and glutamic acid to asparagine and glutamine and converting to a side chain amino group of asparagine and glutamine. Examples include ammonium acetate, ammonium sulfate, ammonium citrate, ammonium chloride, and the like, with ammonium acetate being preferred. The concentration of ammonium salt added to the reaction solution is preferably 5 to 100 mM, more preferably 10 to 50 mM.

At least one of the amino acids in the amino acid mixture and / or at least one of indole, hydrogen sulfide or sulfide salt, and ammonium salt is labeled with a stable isotope. The stable isotope refers to one or a combination of two or more selected from the group consisting of ¹⁷ O, ¹⁸ O, ¹³ C, ¹⁵ N, and ² H. In protein structural analysis, many types of labeled proteins are often required. In such a case, it is desirable to prepare a substrate amino acid mixture in which several kinds of labeled amino acids are combined. Therefore, at least one kind of amino acid and amino acid precursor substance contained in the above amino acid mixture may be labeled, and all kinds may be labeled. The number and type of amino acids to be labeled may be appropriately selected depending on the type of protein to be analyzed, the purpose of analysis, and the like.

2. Method for Producing Stable Isotope Labeled Protein According to the present invention, the above 1. A method for producing a stable isotope-labeled protein using the stable isotope-labeled protein synthesis composition is provided.

  As long as there is an amino acid biosynthetic enzyme group required to synthesize four types of amino acids, L-tryptophan, L-cysteine, L-asparagine, and L-glutamine, from the above composition, The reaction may be carried out in any cell-free protein synthesis system or in a living cell.

  The above amino acid biosynthetic enzymes are tryptophan synthase (EC4.2.1.20) or tryptophanase (EC4.1.99.1) involved in L-tryptophan synthesis, serine o-acetyltransferase (EC2.3.1.30) involved in L-cysteine synthesis. ), Cysteine synthase (EC 4.2.99.8), asparagine synthetase (EC6.3.1.1) involved in L-asparagine synthesis, glutamine synthetase (EC6.3.1.2) related to L-glutamine, etc. It is sufficient that an enzyme group that catalyzes is present in the cell.

2-1. Cell-free protein synthesis system In the present invention, the "cell-free protein synthesis system" refers to a cell-free transcription system that synthesizes RNA using DNA as a template and a cell-free translation system that reads mRNA information and synthesizes proteins on ribosomes. Includes both cell-free transcription and translation systems, as well as cell-free translation systems.

  The “protein” in the present invention refers to a polypeptide having an arbitrary molecular weight composed of a plurality of amino acid residues, and particularly refers to a polypeptide in which a three-dimensional structure is formed.

  Production of a stable isotope-labeled protein by the cell-free protein synthesis system of the present invention is carried out as described above in 1. Except for using the above composition, conventionally known materials for cell-free protein synthesis, that is, cell extracts for cell-free protein synthesis, nucleic acids that serve as templates for encoding target proteins, energy sources (ATP, GTP, creatine phosphate) High energy phosphate bond-containing materials). The cell-free protein synthesis reaction may be referred to JP 2000-175695 A, JP 2005-102513 A, Zubay literature (Geoffrey Zubay, Annual Review of Genetics, 1973 Vol. 7: 267-287). In addition, kits commercially available from Roche Diagnostics, Promega, etc. may be used as appropriate.

  "Cell extract for cell-free protein synthesis" refers to plant cells, animal cells, fungal cells, bacterial cells containing components necessary for translation systems or transcription systems / translation systems involved in protein synthesis such as ribosomes and tRNAs. Refers to the extract. Specific examples include extracts of Escherichia coli, wheat germ, rabbit reticulocytes, mouse L-cells, Ehrlich ascites tumor cells, Hela cells, CHO cells, budding yeast, and the like. The cell extract is prepared according to the method described in Pratt, JM, et al., Transcription and trasnlation-a practical approach (1984), pp. 179-209, and the cells are disrupted with a French press or glass beads to obtain protein components. And a buffer containing several kinds of salts for solubilizing ribosomes, homogenizing, and precipitating insoluble components by centrifugation.

An example of a cell extract that can be suitably used in the present invention is an E. coli S30 cell extract. The S30 cell extract can be prepared by known methods (Geoffrey Zubay, Annual Review of Genetics, 1973 Vol.7: 267-287) from E. coli A19 strain ( rna , met ) BL21 strain codon plus, etc. Commercial products (available from Promega and Novagen) may also be used.

  Further, even when a cell extract for cell-free protein synthesis derived from an organism not having the amino acid biosynthetic enzyme group (for example, derived from a human having no tryptophan synthase) is used, the amino acid biosynthesis is included in the reaction solution. The present invention can also be achieved by adding enzymes.

  The amount of the cell extract for cell-free protein synthesis is not particularly limited, but is preferably in the range of 10 to 40% by weight of the total reaction solution, for example.

  The “nucleic acid encoding the target protein” is not particularly limited as long as it contains the appropriate sequence that encodes the target protein and can be transcribed and / or translated, and includes both RNA and DNA.

  The concentration of the nucleic acid added to the reaction solution for cell-free protein synthesis (hereinafter also referred to as reaction solution) can be appropriately set depending on the protein synthesis activity of the cell extract for cell-free protein synthesis, the type of protein to be synthesized, and the like. However, for example, the final concentration is about 0.5 to 10 μg / mL.

  The energy source in the cell-free protein synthesis system is not particularly limited as long as it is a substance that can be used as an energy source in a living body, but preferably a high energy phosphate bond such as ATP, GTP, creatine phosphate, or phosphoenolpyruvate. The substance which has is mentioned. The concentration of the energy source added to the reaction solution can be appropriately set depending on the protein synthesis activity of the cell extract for cell-free protein synthesis, the type of protein to be synthesized, and the like.

  In the above reaction solution, if necessary, an enzyme involved in ATP regeneration (for example, a combination of phosphoenolpyruvate and pyruvate kinase or a combination of creatine phosphate and creatine kinase), various RNA polymerases (T7, T3, and SP6 RNA polymerase, etc.) and chaperone proteins (for example, DnaJ, DnaK, GroE, GroEL, GroES, HSP70, etc.) having a function of forming a three-dimensional structure of the protein may be added.

  Moreover, a non-protein component can be reinforced to the reaction solution as necessary. A non-protein component is a component that is originally contained in a cell extract for cell-free protein synthesis, but is a component that can be added separately to improve protein synthesis ability, such as tRNA. .

  Furthermore, the reaction solution may contain various additives for protection and / or stabilization of proteins and RNA as necessary. Examples of such additives include ribonuclease (RNase) inhibitors (RNase inhibitors, etc.); reducing agents (dithiothreitol, etc.); RNA stabilizers (spermidine, etc.); protease inhibitors (phenylmethanesulfonyl fluoride (PMSF) ) Etc.). What is necessary is just to set the addition density | concentration to these reaction liquids suitably according to the protein synthesis activity of the cell extract for cell-free protein synthesis | combination to be used, the kind of target protein synthesize | combined, etc.

  For cell-free protein synthesis, any conventionally known batch method or dialysis method may be used.

  For example, when the batch method is used, the reaction solution contains a nucleic acid encoding the target protein, a cell extract for cell-free protein synthesis, the amino acid composition as the constituent material of the target protein, ATP (adenosine 5′-triphosphate ), GTP (guanosine 5'-triphosphate), CTP (cytidine 5'-triphosphate), UTP (uridine 5'-triphosphate), buffer, salts, RNase inhibitor, antibacterial agent, If necessary, RNA polymerase such as T7 RNA polymerase (when DNA is used as a template), tRNA and the like can be contained. In addition, as a ATP regeneration system, a combination of phosphoenolpyruvate and pyruvate kinase or a combination of creatine phosphate and creatine kinase, polyethylene glycol (for example, # 8000), 3 ′, 5′-cAMP, folic acid, a reducing agent (for example, dithiotray) Toll) and the like.

  Here, for example, a buffer such as Hepes-KOH or Tris-OAc can be used as the buffer. Examples of salts that can be used include magnesium acetate, magnesium chloride, potassium acetate, and calcium chloride. Examples of antibacterial agents that can be used include sodium azide and ampicillin.

  The reaction conditions may be appropriately set according to the cell-free protein synthesis cell extract to be used, the target protein to be synthesized, etc., but the temperature is usually 20 to 40 ° C., preferably 23 to 37 ° C., and the time is usually 1 to 5 hours, preferably 3 to 4 hours.

  In addition, when continuously producing the target stable isotope-labeled protein using the dialysis method, the above batch-type reaction solution is used as the dialysis internal solution, and dialyzed against the dialysis external solution 5 to 10 times the volume of the reaction solution. And the produced target protein is recovered from the dialyzed solution. As the dialysis external solution, one obtained by removing the cell extract for cell-free protein synthesis, RNase inhibitor, nucleic acid encoding the target protein, RNA polymerase, pyruvate kinase, creatine kinase, etc. from the dialysis internal solution composition can be used. Accordingly, the dialysis external solution includes, for example, a buffer solution, ATP, GTP, CTP, UTP, salts, the amino acid mixture as a constituent material of the target protein, phosphoenolpyruvate or creatine phosphate as an ATP regeneration system, an antibacterial agent, etc. It only has to be included.

  The fractional molecular weight of the dialysis membrane that separates the dialyzed solution from the dialyzed solution is 3,500 to 100,000, preferably 10,000 to 50,000. Dialysis is usually performed while stirring at 20 to 40 ° C., preferably 23 to 37 ° C., and exchanged with a new external solution, or new nucleic acid may be periodically replenished to the reaction solution.

  Dialysis can be performed by using a dialysis apparatus capable of shaking or stirring (rotating stirring or the like) including and separating the inner liquid and the outer liquid through a dialysis membrane. As an apparatus for small scale reaction, for example, DispoDialyzer (registered trademark) (manufactured by Spectrum) or Slidealyzer (registered trademark) (manufactured by Pierce) can be used. As a large-scale reaction apparatus, a Spectra / Por (registered trademark) dialysis tube (manufactured by Spectrum) or the like can be used. The shaking speed or stirring speed is low, for example, 100 to 200 rpm, and the reaction time can be appropriately selected while monitoring the production of the target protein.

  Purification methods for the synthesized stable isotope labeled protein include, for example, ammonium sulfate or acetone precipitation, acid extraction, anion or cation exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, gel filtration chromatography, hydroxyapatite, isoelectric Point chromatography, chromatofocusing and the like can be mentioned, and purification can be carried out by these methods alone or in appropriate combination depending on the properties of the protein. Alternatively, an affinity purification method can be used in which a peptide sequence called a tag is added to the protein in advance and the tag is specifically recognized and adsorbed. This purification method is particularly preferable for obtaining a highly pure protein. Although it does not specifically limit as the said tag, A histidine tag, a GST tag, a maltose binding tag, etc. are common.

  Identification and quantification of the stable isotope-labeled protein synthesized and purified as described above is carried out by comparing with a standard sample as required by activity measurement, immunological measurement, spectroscopic measurement, amino acid analysis, etc. be able to.

2-2. Live cell synthesis system The method of performing stable isotope-labeled protein in living cells may be a method of directly adding the composition for stable isotope-labeled protein synthesis to a culture medium for culturing live cells, or A method in which live cells cultured in advance are collected, washed, and reacted with a buffer solution to which the composition for synthesizing stable isotope-labeled protein is added (see T. Yamasaki J. Am. Chem. Soc. 1997, 199 p872-880). Any method of) may be used.

  Similarly, when a living cell is used, it may be a cell derived from an organism having the above amino acid biosynthetic enzyme group, and examples thereof include Escherichia coli and yeast.

  However, in order to keep the hydrogen sulfide generated from the indole and sodium sulfide to be added at a low concentration that does not show cytotoxicity, it is desirable to add them intermittently while monitoring their concentrations.

  When producing a stable isotope-labeled protein in a living cell, first, a nucleic acid encoding the target protein or an appropriate plasmid incorporating the nucleic acid is introduced into the living cell to obtain a recombinant cell. Next, the obtained recombinant cells are cultured in a culture solution containing the above composition, or the cultured cells obtained by culturing the obtained recombinant cells in a normal culture solution are collected and washed, and then the above composition is prepared. Add to the buffer containing the product and incubate.

  The target stable isotope labeled protein can be obtained by collecting from the culture. Here, the “culture” means not only the culture supernatant but also any cultured cells or cultured cells, or crushed cells or cells.

  When culturing a recombinant cell, it is carried out according to the usual method used for culturing the cell. If the cell is a microorganism such as Escherichia coli or yeast, a natural medium that contains a carbon source, a nitrogen source, an inorganic salt, etc. that can be assimilated by the microorganism and can efficiently culture the cell. Any of synthetic media may be used. Examples of the carbon source include carbohydrates such as glucose, fructose, sucrose, and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol. Examples of the nitrogen source include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salts of organic acids such as ammonium phosphate or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor, and the like. Examples of the inorganic substance include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate. The culture is usually performed at 37 ° C. under aerobic conditions such as shaking culture or aeration and agitation culture. The pH is adjusted using an inorganic or organic acid, an alkaline solution, or the like. During culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as necessary.

  When the cultured recombinant cells are collected by a known method and added to the buffer containing the above composition, examples of the buffer include minimal inorganic medium, Hepes buffer, Tris buffer, and phosphate buffer. Can be used.

  When the target protein is produced in cells or cells, the target protein is collected by crushing the cells or cells by performing ultrasonic treatment, repeated freeze-thawing, homogenizer treatment, and the like. When the target protein is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like. Thereafter, by using general biochemical methods used for protein isolation and purification, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc. alone or in appropriate combination, From the above, the protein of the present invention can be isolated and purified.

3. Reagent kit for synthesis of stable isotope labeled protein The “stable isotope-labeled protein synthesis composition” is provided in the form of a stable isotope-labeled protein synthesis kit together with other components necessary for protein production by a cell-free protein synthesis system or a living cell synthesis system. be able to.

  For example, when stable isotope-labeled protein synthesis is carried out in a cell-free protein synthesis system, the stable isotope-labeled protein synthesis kit of the present invention comprises the aforementioned composition for stable isotope-labeled protein synthesis and cells for cell-free protein synthesis What is necessary is just to contain the extract at least. In addition, the kit can contain ribonucleotides such as ATP, GTP, CTP, and UTP, and a buffer for adjusting the pH of the substrate solution.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but these examples do not limit the present invention.

(Example 1) Cell-free protein synthesis using a mixture of 19 amino acids (without L-tryptophan) and indole
Cell-free protein synthesis with 19 kinds of amino acid mixtures excluding L-tryptophan and indole was performed by dialysis. The compositions of the reaction solution (inner solution) and the dialysis outer solution other than amino acids and amino acid precursors are shown in Table 1 and Table 2, respectively.

  The reaction was performed by dialysis of a reaction solution (inner solution) having the composition shown in Table 1 overnight at 30 ° C. against an external dialysis solution having the composition shown in Table 2. The reaction scale was 30 μl of reaction solution (inner solution) and 300 μl of dialysis solution. E. coli S30 extract was prepared from E. coli BL21codon plus strain according to the method of Zubay et al. (Annu. Rev. Geneti., 7, 267-287, 1973). In order to quantitatively show the amount of protein synthesis, chloramphenicol aminotransferase (CAT), which can easily quantify proteins from specific activity, was employed. PK7-CAT (see Kim et al., Eur. J. Biochem. 239, 881-886, 1996) was used as the CAT expression vector. The synthesized CAT protein was quantified according to the method of Shaw et al. (See Methods Enzymol. 735-755, 1975). That is, acetylation of chloramphenicol by CAT using acetyl coenzyme A and chloramphenicol as substrates, and the resulting reduced coenzyme A was converted to 5,5′-dithiobis-2-nitrobenzoic acid (DNTB). The color development was quantified using The activity of CAT was quantified from the increase in absorbance per unit time at 37 ° C. and 412 nm, and the amount of CAT protein was determined using this as an index.

  For the reaction solution (Table 1), as amino acids other than L-tryptophan, a mixture of 16 types of amino acids (L-alanine, L-arginine, L-aspartic acid, L -Glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tyrosine, L-valine) ( Purchased from Taiyo Nippon Sanso Co., Ltd.) at a final concentration of 5 mg / ml, L-asparagine at a final concentration of 2 mM, and L-glutamine and L-cysteine at a final concentration of 1 mM. Further, indole as a tryptophan precursor (substrate) was added to the reaction solution so that the final concentration was 0.5 mM or 1.0 mM. In addition, as a control, CAT synthesis was simultaneously performed in the absence of indole and in the condition where L-tryptophan was added to a final concentration of 1 mM.

  As a result, as shown in FIG. 1, L-tryptophan and indole were not added to the reaction system (in FIG. 1, 19 types of amino acids, background), and almost no CAT was synthesized. Synthesized. In addition, by adding 0.5 mM of pyridoxal 5'-phosphate (PLP), a coenzyme for tryptophan synthase, at the final concentration, CAT was synthesized in the same amount as when 20 amino acids were added.

(Example 2) Cell-free protein synthesis using a mixture of 19 amino acids (without L-cysteine) and sodium sulfide Cell-free protein synthesis using a mixture of 19 amino acids excluding L-cysteine and sodium sulfide was carried out in the same manner as in Example 1. The dialysis method was used. The reaction solution (inner solution) composition and the dialysis outer solution composition other than amino acids and amino acid precursors, and the reaction conditions (scale, temperature, time) are the same as in Example 1. For the reaction solution (Table 1), as amino acids other than L-cysteine, a mixture of 16 kinds of amino acids (L-alanine, L-arginine, L-aspartic acid, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tyrosine, L-valine) (Purchased from Taiyo Nippon Sanso Co., Ltd.) was added to a final concentration of 5 mg / ml, L-asparagine to a final concentration of 2 mM, and L-glutamine and L-tryptophan to a final concentration of 1 mM. In addition, sodium sulfide (Na ₂ S) as a precursor (substrate) of L-cysteine was added to the reaction solution so that the final concentration was 0.5 mM or 1.0 mM. Further, as a control, CAT synthesis was simultaneously performed under the condition where sodium sulfide was not added and under the condition where L-cysteine was added to a final concentration of 1.0 mM.

  As a result, as shown in FIG. 2, CAT was hardly synthesized in the reaction system to which L-cysteine and sodium sulfide were not added (in FIG. 2, 19 kinds of amino acids, background), but sodium sulfide was added. As a result, CAT was synthesized. Further, as in Example 1, the amount of synthesis further increased by adding 0.5 mM of PLP, which is a coenzyme of cysteine synthase, at a final concentration. When sodium sulfide was added at a final concentration of 1 mM and PLP was added at a final concentration of 0.5 mM, over 80% of CAT protein was synthesized when 20 types of amino acids were added.

(Example 3) A mixture of 18 kinds of amino acids (without addition of L-cysteine and L-tryptophan) or a mixture of 16 kinds of amino acids (without addition of L-asparagine, L-glutamine, L-cysteine and L-tryptophan) and sodium sulfide Cell-free protein synthesis by indole
(i) Cell-free protein synthesis with a mixture of 18 amino acids excluding L-cysteine and L-tryptophan, sodium sulfide and indole, (ii) Excluding L-asparagine, L-glutamine, L-cysteine and L-tryptophan16 Cell-free protein synthesis with a mixture of various amino acids, sodium sulfide, and indole was performed by dialysis in the same manner as in Example 1. The reaction solution (inner solution) composition and the dialysis outer solution composition other than amino acids and amino acid precursors, and the reaction conditions (scale, temperature, time) are the same as in Example 1.

For the reaction solution (Table 1), as amino acids other than L-cysteine and L-tryptophan, a mixture of 16 types of amino acids (L-alanine, L-arginine, L -Aspartic acid, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tyrosine, L-valine (purchased from Taiyo Nippon Sanso Co., Ltd.) at a final concentration of 5 mg / ml. In the case of (i) synthesis, L-asparagine is 2 mM in the final concentration and L-glutamine is 1 mM in the final concentration. Was added to the reaction solution. In addition, sodium sulfide (Na ₂ S), which is the precursor (substrate) of L-cysteine, is 0.5 mM in the final concentration, and indole, which is the precursor (substrate) of L-tryptophan, at the final concentration. It added so that it might become 0, 0.1, 0.2, 0.3, 0.5, 1.0, 1.5 mM. Furthermore, 0.5 mM of PLP, which was effective in improving the CAT synthesis amount in Examples 1 and 2, was added at the final concentration. As a control, CAT synthesis was simultaneously performed under the condition where L-cysteine and L-tryptophan were added to a final concentration of 1 mM.

  As a result, as shown in FIG. 3, in the reaction system to which no indole was added, CAT was hardly synthesized, but CAT was synthesized by adding indole. The amount of synthesis was the highest when the final concentration of indole was 0.2 mM. In the case of 16 types of amino acids, it was confirmed that CAT was synthesized although the synthesis amount remained at about 60% compared to the case of 18 types of amino acids. By optimizing ammonium ions in the reaction system, the synthesis amount is considered to be further improved.

(Example 4) nitrogen isotopes ⁽¹⁵ N) Synthesis of labeled protein (1) nitrogen isotopic cell-free protein synthesis ⁽¹⁵ N) labeled protein synthesis
16 kinds of ¹⁵ N labeled amino acid mixture (L-alanine- ¹⁵ N, L-arginine- ¹⁵ N, L-aspartic acid- ¹⁵ N, L-glutamic acid- ¹⁵ N, glycine- ¹⁵ N, L-histidine- ¹⁵ N, L-isoleucine- ¹⁵ N, L-leucine- ¹⁵ N , L-lysine- ¹⁵ N, L-methionine- ¹⁵ N, L-phenylalanine- ¹⁵ N, L-proline- ¹⁵ N, L-serine- ¹⁵ N, L-threonine- ¹⁵ N, L-tyrosine- ¹⁵ N, L- valine - ¹⁵ N), sodium sulfide (Na ₂ S), and indol as amino acid substrate, in place of the CAT expression vector as the template DNA (expression plasmid) shown in table 1 Ras protein (Yamasaki J Biomol NMR. 1992 Jan; 2 (1): 71-82) circular plasmid DNA (final concentration 2 μg / ml), cell-free as in Example 1 except that ammonium acetate- ¹⁵ N is used instead of ammonium acetate Protein synthesis was performed by dialysis. The reaction solution (inner solution) composition, the dialysis outer solution composition, and the reaction conditions (temperature and time) other than the amino acid and the amino acid precursor are the same as in Example 1. The reaction solution (inner solution) was 3 ml, and the outer dialysis solution was 30 ml.

To the reaction solution (Table 1), a nitrogen isotope ( ¹⁵ N) amino acid mixture was added to a final concentration of 5 mg / ml, indole to a final concentration of 0.2 mM, and sodium sulfide to a final concentration of 0.5 mM. In addition, PLP as a coenzyme was added to a final concentration of 0.5 mM. As a control, cell-free protein synthesis using 20 types of ¹⁵ N-labeled amino acids as substrates was also performed. After the synthesis was completed, the reaction solution in the dialysis membrane was collected in a centrifuge tube.

(2) Purification of nitrogen isotope ( ¹⁵ N) -labeled protein The Ras protein synthesized and recovered by the above method was purified by a purification method utilizing the affinity between histidine and cobalt ions. The operation was performed at 4 ° C. 3 ml of the cell-free protein synthesis reaction solution was diluted with 2.6 ml of a buffer solution (20 mM Tris hydrochloride (pH 8.0) / 1 M sodium chloride), and centrifuged (2,000 rpm, 5 minutes) to remove the precipitate. To the resulting supernatant, 3.2 ml of TALON resin (BD Biosciences clontech) was added and mixed to adsorb the protein. The mini-column was gently filled with the mixed solution of the protein solution and the resin, and the resin and the liquid were separated by filtration. After washing the column with about 5 times the column volume of washing buffer, add 4 ml of elution buffer (20 mM Tris hydrochloride (pH 8.0) / 30 mM sodium chloride / 500 mM imidazole) to the resin. The Ras protein adsorbed on was eluted. In order to cleave the unnecessary histidine tag at the N-terminus of the eluted Ras protein, it was concentrated to about 500 μl using an ultrafiltration device (Vivapin 2; Sartorius), added with 8 μg / ml TEV protease, and 4 ° C. The cleavage reaction was performed overnight. The solution after completion of the reaction was put into the ultrafiltration device again, and 2 ml of a buffer solution (20 mM Tris hydrochloride (pH 8.0) / 1 M sodium chloride) was added and concentrated to 200 μl. This was repeated several times to exchange the buffer solution. The washing buffer was further added to the Ras protein solution in which the solvent was replaced with the washing buffer to make 3.5 ml, and 1.6 ml of TALON resin was added thereto to adsorb the cleaved histidine tag to the resin. The minicolumn was filled with a solution containing the resin, and the fraction passed through was collected to obtain purified Ras protein. The yield was about 3.1 mg when synthesized with 16 amino acids and 3.8 mg when synthesized with 20 amino acids, and the yield of Ras protein synthesized with 16 amino acids according to the method of the present invention was synthesized with 20 amino acids. Although the yield was slightly reduced as compared with the case, an amount of Ras protein sufficient for NMR measurement could be synthesized.

(3) solvent and ¹⁵ N-labeled Ras proteins prepared purified NMR measurement sample suitable for the NMR measurement (20mM sodium phosphate (pH6.5) / 100mM NaCl / 5mM MgCl 2 / 5mM d-DTT / 0.01% NaN 3) Then, the solvent was exchanged again using an ultrafiltration apparatus. Heavy water was added to the solvent-substituted protein solution at 10% to prepare a sample for NMR measurement.

(4) NMR measurement
¹⁵ N substituted with solvent for NMR measurement The labeled Ras protein solution is placed in a symmetric micro NMR sample tube (Shigemi, φ5 mm) and ¹ H- ¹⁵ N at a temperature of 25 ° C. with a 700 MHz NMR apparatus (DRX; Bruker). -Measured 2D HSQC (hereinafter abbreviated as ¹⁵ N-HSQC). The results of NMR measurement ( ¹⁵ N-HSQC spectrum) are shown in FIG.

  In the Ras protein synthesized using 16 types of amino acids and other amino acid precursors as amino acid sources, unlabeled indole was used as the tryptophan precursor, indicating that the side chain (indole ring) of tryptophan was not labeled (Fig. 4: Below). The other NH signals were confirmed to be equivalent to Ras protein synthesized with 20 amino acids.

(Example 5) Synthesis of oxygen isotope ( ¹⁸ O) labeled protein (1) Oxygen isotope ( ¹⁸ O) labeling of amino acids (16 types) As a method of labeling oxygen of the carboxyl group of amino acids with oxygen isotopes, Murphy RC, Clay KL., 1979, Synthesis and back exchange of ¹⁸ O-labeled amino acids for use as internal standards with mass spectrometry. The method described in Biomed Mass Spectrom 6: 309-314 was used.

16 commercially available amino acids (L-alanine, L-arginine, L-aspartic acid, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tyrosine, L-valine) are mixed so that the composition ratio is the same as that of the amino acid mixture of cyanobacteria shown in Table 3 below. A mixture of amino acids was made.

The amino acid mixture was weighed in 0.257 g (A) and 0.259 g (B), and each was transferred to a 10 ml eggplant flask. To A, 0.35 hydrochloric acid ¹⁸ O-labeled water ( ¹⁸ O concentration: 95 atom%) prepared with concentrated hydrochloric acid (4.25 g) was added to B, and 0.3N hydrochloric acid aqueous solution (unlabeled water) (4.02 g) was added and completely dissolved. Each was covered and placed in an oil bath and heated at 100 ° C. for 16 hours under a nitrogen stream. After completion of the reaction, the mixture was cooled to room temperature and completely dried using a rotary evaporator to remove water and hydrochloric acid. Distilled water was added to the dried product, adjusted to pH 7.5 with 5M potassium hydroxide, and each was added with distilled water to 5 ml to prepare oxygen isotope ( ¹⁸ O) labeled amino acid mixture (16 types).

(2) oxygen isotope by cell-free protein synthesis method ⁽¹⁸ O) of oxygen isotopes created in the labeled protein synthesis amino acid substrates (1) ⁽¹⁸ O) using labeled amino acid mixture, template DNA shown in Table 1 (expressed Example 1 except that expression plasmid DNA containing the ubiquitin-binding domain (UBA) base sequence of human isopeptidase 5 (EC3.1.2.15) shown in SEQ ID NO: 1 was used as the plasmid) instead of the CAT expression vector In the same manner, cell-free protein synthesis was performed by dialysis. The reaction solution (inner solution) composition and the dialysis outer solution composition other than amino acids and amino acid precursors, and the reaction conditions (scale, temperature, time) are the same as in Example 1.

To the reaction solution (Table 1), an oxygen isotope ( ¹⁸ O) labeled amino acid mixture and an unlabeled amino acid mixture were added to a final concentration of 5 mg / ml. Indole and sodium sulfide were added to a final concentration of 1 mM. In addition, as a control, UBA protein was also used under the conditions where unlabeled L-asparagine was added to a final concentration of 2 mM, and unlabeled L-glutamine, unlabeled L-tryptophan, and unlabeled L-cysteine were each added to a final concentration of 1 mM. Synthesis was performed.

(3) Purification of Oxygen Isotope ( ¹⁸ O) Labeled Protein The UBA protein synthesized as described above was purified by a purification method using the affinity between histidine and cobalt ions. Collect 30 μl of the cell-free protein synthesis reaction solution in a 0.6 ml centrifuge tube, dilute with 30 μl of buffer solution (20 mM Tris hydrochloride (pH 8.0) / 1 M sodium chloride), and centrifuge (4,000 rpm, 5 minutes). To remove the precipitate. TALON resin (BD Biosciences clontech) was added to 40 μl of the obtained supernatant and mixed to adsorb the protein. After removing the supernatant by centrifugation using a filter plate, 150 μl of the above buffer solution was added to the obtained resin, and the mixture was further centrifuged to wash the resin. To elute the synthetic UBA protein from the resin, add 100 μl of elution buffer (20 mM Tris hydrochloride (pH 8.0) / 300 mM sodium chloride / 500 mM imidazole) to the resin, mix, and centrifuge to elute the purified UBA protein. A liquid was obtained.

(4) oxygen isotope ⁽¹⁸ O) desalting and mass spectrometry purified oxygen isotope labeled protein ⁽¹⁸ O) in order to confirm the presence or absence of the mass increase due to oxygen isotope label of the labeled UBA proteins, mass spectrometry went. As a pretreatment for mass spectrometry, the purified protein was desalted with Zip tip C18 (Waters). For mass analysis accuracy, by adding myoglobin as an internal standard in the sample and desalted purified UBA protein samples, MALDI-TOF MS; mass of oxygen isotope by (Voyager Applied Biosystems) ⁽¹⁸ O) labeled protein Was measured. The average molecular weight of the unlabeled UBA protein was 10390.9 Da. The mass measurement result by TOF-MS is shown in FIG. As a result, it was confirmed that the UBA protein using the ¹⁸ O-labeled amino acid mixture had an increase in average mass that was probably due to oxygen isotope labeling.

  According to the present invention, it is possible to produce a stable isotope-labeled protein at a low cost, which can contribute to technical fields such as protein tertiary structure analysis.

  All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (6)

  (A) L-alanine, L-arginine, L-aspartic acid, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, A mixture of 16 amino acids of L-serine, L-threonine, L-tyrosine, L-valine, (B) indole, (C) hydrogen sulfide or sulfide salt, and (D) ammonium salt, and A stable isotope-labeled protein, wherein at least one of amino acids in the amino acid mixture and / or at least one of indole, hydrogen sulfide or sulfide salt, and ammonium salt is labeled with a stable isotope Composition for synthesis. The stable isotope labeled protein synthesis according to claim 1, wherein the stable isotope is one or a combination of two or more selected from the group consisting of ¹⁷ O, ¹⁸ O, ¹³ C, ¹⁵ N, and ² H. Composition.   A reagent kit for synthesizing stable isotope-labeled proteins, comprising the composition according to claim 1 or 2.   A method for producing a stable isotope-labeled protein, comprising synthesizing a stable isotope-labeled protein using the composition according to claim 1.   The production method according to claim 4, wherein the stable isotope-labeled protein is synthesized in a cell-free protein synthesis system.   The production method according to claim 4, wherein the synthesis of the stable isotope-labeled protein is performed in a living cell.

Images