JP4193249B2 - Method for producing C-terminally labeled protein - Google Patents

Description

【配列表フリーテキスト】
配列番号１−６：合成ＤＮＡ
【図面の簡単な説明】
【図１】ピューロマイシンおよびその誘導体の化学構造。Iはピューロマイシン(puromycin)、IIはフルオレセニルピューロマイシン(Fluorpur)である。
【図２】β-ラクタマーゼの遺伝子DNAの構築図。Aは終止コドンのない遺伝子DNA、Bは終止コドンのある遺伝子DNA である。
【図３】β-ラクタマーゼのC末端へのFluorpurの結合を示すポリアクリルアミドゲル電気泳動写真(A)とそれをSYPRO Orangeで蛍光染色したポリアクリルアミドゲル電気泳動写真(B)。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for efficiently labeling the C-terminus of a protein with a labeling reagent. The C-terminal labeling method of proteins with the labeling reagent of the present invention is effective for detection and identification of proteins expressed in various cell-free translation systems and living cells, and can be used for functional analysis of genes accumulated by genome analysis. It provides an extremely effective means for efficient and automated protein identification, such as nucleic acid-protein interaction and protein-protein interaction, particularly for identification of proteins that retain the function to function.
[0002]
[Prior art]
For labeling proteins expressed in cell-free translation systems and living cells, a radioactive labeling method that incorporates amino acids labeled with radioactive elements such as ³⁵ S, ³ H, and ¹⁴ C into translation products is common. . In this case, a special facility is required for safety management in order to use radioactivity. For this reason, the following method is known as a method not using a radioactive compound. According to this method, biotin is first covalently bonded to the ε-amino group of the amino acid lysine, and this is ester-linked to a tRNA having an anticodon of lysine (biotin-lysine-tRNA), and cell-free. Input into the translation system and biotinylate the translation product. The translation product is transferred to a membrane after electrophoresis, and the translation product is chemiluminescent with alkaline phosphatase using a fusion protein of alkaline phosphatase and streptavidin. A translation product is identified from this using an X-ray film or the like (Promega, (1993) Technical Bulletin, No. 182, p2). This method has the disadvantage that the synthesized biotin-lysine-tRNA is very unstable (6 months at -70 ° C.) and is expensive. Furthermore, the prescription until identification is complicated and takes time. Therefore, this method is extremely disadvantageous for automation for processing a large amount of samples. Furthermore, the translated protein may have a function and structure that is different from the original because a plurality of lysine side chains are modified with biotin.
[0003]
[Problems to be solved by the invention]
The problems of the present invention are as follows: 1) efficient, 2) simple, 2) economical, and stable labeling reagent for a long period of time in labeling of translated proteins in cell-free translation systems and living cells. 4) The function and structure of the translation product are not affected. 5) The present invention is to provide a method for producing a protein labeled with a C-terminus, which satisfies conditions such as safety.
[0004]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the present inventors have found that a labeling reagent comprising a label part composed of a labeling substance and an acceptor part composed of a compound having the ability to bind to the C-terminus of a protein. When protein synthesis is performed in a cell-free translation system or in a living cell using a product transcribed from DNA consisting of a coding region with the termination codon deleted under the control of the promoter region as a template, the C-terminal of the protein The present inventors have found that the C-terminus of a high molecular weight protein can be efficiently labeled without impairing the function of the present invention, thereby completing the present invention.
That is, the present invention
(1) from the consisting labeling substance label part has a puromycin or 3 '-N-amino acyl puromycin aminonucleoside of chemical structure, the acceptor portion made of a compound having the ability to bind to the C-terminus of the translated protein Protein synthesis in a cell-free translation system or living cell system using as a template a product transcribed from a coding region with a coding region deleted from the stop codon under the control of the promoter region. A method for producing a protein labeled at the C-terminus, comprising:
(2) The method according to (1) above, wherein the coding region is DNA having a length corresponding to 50-3,000 amino acid residues,
(3) The method according to (1) or (2) above, wherein the label part of the labeling reagent is a fluorescent substance, a radioactive substance, or a non-radioactive labeling substance,
( 4 ) The method according to any one of (1) to (3) above, wherein the cell-free translation system uses wheat germ extract or rabbit reticulocyte extract,
Exist.
A feature of the present invention is that a product (processed mRNA) transcribed from DNA consisting of a coding region in which a stop codon is deleted under the control of a promoter region in a cell-free translation system or a living cell system is added as a template, and the final concentration When protein synthesis is performed in the presence of a labeling reagent such as 15-50 μM puromycin or a puromycin derivative, the labeled substance efficiently binds to the C-terminus of the translated protein, and the high molecular weight is labeled at the C-terminus. It is that the protein of is obtained. A compound in which a fluorescent substance such as fluorescein is chemically bound to puromycin binds to the C terminus of the translated protein without impairing the function of the C terminus of the protein, as with puromycin. It was found that identification of That is, after adding a labeling reagent such as fluorescent puromycin to a cell-free translation system, the reaction product is electrophoresed, and the gel is read as it is with a fluorescent image analyzer, whereby the translated protein can be easily identified.
[0005]
DETAILED DESCRIPTION OF THE INVENTION
The protein labeled with the C-terminus of the protein of the present invention is obtained by adding a processed DNA transcription product (processed mRNA) to a cell-free translation system or a living cell system as a template, a label portion comprising a labeling substance, It is produced by performing protein synthesis in the presence of a labeling reagent composed of an acceptor moiety composed of a compound having the ability to bind to the C-terminus.
[0006]
The product (processed mRNA) transcribed from the DNA consisting of the coding region with the stop codon deleted under the control of the promoter region (processed mRNA) is RNA from the DNA consisting of the promoter region and the coding region with the stop codon deleted. It is obtained by transcription using a polymerase. The length of the coding region is preferably DNA having a length corresponding to 50-3,000 amino acid residues. That is, according to the method of the present invention, even a high molecular weight protein can efficiently label its C-terminus, which is very useful for identification of proteins obtained by genome analysis and functional analysis thereof.
[0007]
In the method of the present invention, the C-terminus of a protein is labeled with a labeling reagent. The labeling reagent is composed of a label part and an acceptor part. The label part is usually selected from a fluorescent substance, a radioactive substance and a non-radioactive labeling substance. In addition to the fluorescein series, the label fluorescent substance has a free functional group (for example, carboxyl group, hydroxyl group, amino group, etc.) and can be linked to nucleotide derivatives such as puromycin or puromycin-like compounds via a spacer. Various fluorescent dyes (for example, rhodamine series, eosin series, NBD series, etc.) may be used.
[0008]
Other labeling parts include radiolabeled substances such as ³³ P, ³² P and ³⁵ S, coenzymes such as biotin, proteins, peptides, saccharides, lipids, dyes, non-radioactive labels such as polyethylene glycol, or If it is a compound which can be labeled, the kind and magnitude | size of the compound will not ask | require.
[0009]
As the acceptor part which is another component constituting the labeling reagent, a nucleic acid or a nucleic acid derivative is usually used. As the nucleic acid derivative, a compound in which a nucleic acid and an amino acid or a substance having a chemical structure skeleton similar to an amino acid is chemically bonded can be used. Typical compounds include puromycin having an amide bond, 3'-N-aminoacylpuromycin aminonucleoside (PANS-amino acid), for example, PANS-Gly having an amino acid moiety of glycine , PANS-Val for valine, PANS-Ala for alanine, and other PANS-all amino acid compounds corresponding to all amino acids. In addition, 3'-Aminoacyladenosine aminonucleoside (AANS-amino acid, 3'-Aminoacyladenosine aminonucleoside, connected by an amide bond formed as a result of dehydration condensation of the amino group of 3'-aminoadenosine and the carboxyl group of amino acid as a chemical bond For example, AANS-Gly whose amino acid part is glycine, AANS-Val of valine, AANS-Ala of alanine, and other AANS-all amino acid compounds corresponding to all amino acids can be used. Nucleosides or nucleotide-amino acid ester bonds can also be used. In addition, if a substance having a chemical structure skeleton and base similar to nucleic acid or a substance having a chemical structure skeleton similar to amino acid can be chemically bonded, all the compounds bonded in this way are used. it can.
[0010]
Puromycin (compound I in FIG. 1) is a bacterium (Nathans, D. (1964) Proc. Natl. Acad. Sci. USA, 51, 585-592; Takeda, Y. et al. (1960) J. Biochem. 48,169-177) and animal cells (Ferguson, JJ (1962) Biochim. Biophys. Acta 57,616-617; Nemeth, AM & de la Haba, GL (1962) J. Biol. Chem. 237, 1190-1193) It is known to inhibit synthesis.
Since the structure of puromycin is similar to that of aminoacyl-tRNA, it enters the A site on the ribosome, reacts with the peptidyl tRNA present at the P site, and is released from the ribosome as peptidylpuromycin (Harris, RJ ( 1971) Biochim. Biophys. Acta 240, 244-262).
[0011]
In protein synthesis systems, it is known that protein synthesis stops when cleaved mRNA without a stop codon is used as a template. Thus, when there is no codon corresponding to mRNA, aminoacyl-tRNA and termination factor are not catalyzed by the peptide transfer reaction even if they enter the A site on the ribosome. On the other hand, it was found that puromycin and the derivatives of the present invention were catalyzed by the peptide transfer reaction of ribosome at the A site on the ribosome and could efficiently bind to the C-terminus of the protein even in such a state.
[0012]
In order to confirm this, an mRNA having a stop codon and an mRNA having no stop codon were prepared and cell-free together with a fluorescent puromycin derivative such as fluorescylpuromycin (Fluorpur) (compound II in FIG. 1). In addition to the translation system, it is necessary to examine the efficiency of fluorescent labeling of the C-terminus of proteins.
[0013]
In order to prepare mRNAs of β-lactamase (molecular weight 32 kDa) with a standard molecular weight without β-lactamase (molecular weight 32 kDa) and some, the T7 promoter and Kozak sequence from the 5 ′ side as shown in FIG. (Kozak sequence), β-lactamase coding region, and the gene DNAs (A) and (B) without a stop codon.
[0014]
As a fluorescent puromycin derivative, fluorescein is selected as the label part, puromycin is selected as the acceptor part, and a fluorescent labeled compound in which both are linked by a chemical bond, for example, Fluorpur (compound II in FIG. 1) is chemically synthesized. did.
[0015]
In eukaryotic cell-free transcription and translation systems, such as the translation system of rabbit reticulocyte lysate and wheat germ extract, the above T7 promoter, Kozak sequence, β-lactamase coding region, and termination When transcripts of DNA with or without codons (processed mRNA) are added as templates, protein synthesis is performed in the presence of Fluorpur, and the fluorescent labeling of the protein is examined, the C-terminal of the full-length protein of β-lactamase Fluorpur was clearly bound at a concentration of 16 μM (FIG. 3A). The fluorescent labeling of the full-length protein of β-lactamase by Fluorpur was confirmed in the cell-free transcription translation system of E. coli. In particular, when a cell-free translation system of wheat germ extract is used, mRNA without a stop codon (lane 1 in FIG. 3) is labeled about 10 times as much as mRNA with a stop codon (lane 3 in FIG. 3). It was confirmed that the conversion efficiency increased.
[0016]
【Example】
The present invention will be described more specifically with reference to the following examples. However, the following examples should be regarded as an aid for obtaining specific recognition about the present invention, and the scope of the present invention is not limited by the following examples. It is not limited.
[0017]
Example 1
C-terminal fluorescent labeling of β-lactamase using cell-free translation system of eukaryotic cells (wheat germ extract, rabbit reticulocyte extract) <1> Construction of transcriptional DNA and mRNA preparation material: β-lactamase gene PBR322 plasmid (Sutcliffe, JG (1978) Proc. Natl. Acad. Sci. USA, 75, 3737-3741) is provided by Mr. Masamichi Ishizaka (Kyushu University, Mitsubishi Chemical Life Science Institute). DNA containing T7 promoter and Kozak consensus sequence of E. coli pAR vector (Rosenberg, AH et.al. (1987) Gene 56, 125-135) was synthesized by Nippon Flour Co., Ltd. DNA primers for PCR were synthesized by Espec Oligo Service, and DNA / RNA primers were synthesized by Nippon Genset. Commercially available enzymes and reagents were used: RNA-degrading enzyme Ribonuclease A (Sigma); Nucleic acid modifying enzyme T4 DNA Polynucleotide Kinase (NEB), T4 DNA ligase (NEB); Thermostable DNA synthase Gold Taq. Polymerase ( Perkin-Elmer); RNA synthase kit Ribomax Large Scale RNA Production System (Promega); Cap analog RNA capping Analog (Gibco BRL); Cell-free translation system kit: Rabbit Reticulocyte Lysate Systems, Nuclease Treated, Promega ), Wheat Germ Extract, Promega; Electrophoretic reagents Acrylamide, acrylamide-bis, SDS, etc. are all made by Nacalai Tesque. Primer Remover was purchased from Edge.
[0018]
Method: DNA having a DNA sequence (T7 promoter sequence) recognized by E. coli virus T7 RNA polymerase with high transcription efficiency and a sequence (Kozak consensus sequence) easily recognized by eukaryotic ribosomes during translation (Fig. 2) ) Was constructed as follows. First, a region having the above sequence and a region having a β-lactamase gene are prepared independently. A single-stranded DNA (SEQ ID NO: 1) containing a T7 promoter sequence and a Kozak consensus sequence is chemically synthesized, and PCR is performed using a DNA primer (SEQ ID NO: 2) and a DNA / RNA primer (SEQ ID NO: 3). On the other hand, the pBR322 plasmid carrying the β-lactamase gene is used as a template and PCR is carried out with a DNA / RNA primer (SEQ ID NO: 4) and a DNA primer (SEQ ID NO: 5) to amplify the β-lactamase gene DNA region. In accordance with the RRR method (Nishigaki, K. et al. (1995) Chem. Lett. 131-132), ribonuclease A was added to each PCR reaction solution and reacted at 60 ° C. for 30 minutes, whereby DNA / RNA primer Cleavage of the phosphodiester bond on the 3 ′ side of the RNA of (SEQ ID NO: 3 and SEQ ID NO: 4) creates a protruding end. After phenol extraction, the primer and the cleaved DNA fragment are removed with a primer remover and ethanol precipitated. After drying, dissolve in T4 DNA ligase buffer, add T4 DNA polynucleotide kinase (Polynucleotide Kinase), react at 45 ° C for 30 minutes, then gradually lower the temperature to 16 ° C over 30 minutes before T4 DNA ligase. And the above two DNA regions were combined. A part of this reaction solution was collected and amplified again by PCR using DNA primers (SEQ ID NO: 2 and SEQ ID NO: 5) to prepare (A) in FIG. 2 for transcription.
[0019]
On the other hand, a transcriptional DNA comprising a β-lactamase gene having a stop codon (B in FIG. 2) is obtained by using another DNA primer (SEQ ID NO: 6) instead of the DNA primer (SEQ ID NO: 5) in the above method. Created.
The two DNAs prepared by the above method were transcribed using an RNA synthesis kit Ribomax Large Scale RNA Production System (Promega). In order to increase the synthesis efficiency, cap analog RNA capping Analog (Gibco BRL) was used to modify the 5 ′ side of mRNA. Ethanol precipitation was performed using a primer remover to remove the cap analog and excess NTP.
[0020]
<2> Preparation material of fluorescence leveling reagent Fluorpur: Puromycin is from Sigma, fluoredite (6-N-carboxy-di-O-pivaloyl-fluorescein-hexyl-O- (2-cyanoethyl)-(N, N'-diisopropyl) -phosphoramidite) was purchased from Nippon Perceptive, tetrazole from Nippon Millipore, and chromatographic silica gel from Merck.
[0021]
Method: Puromycin (26 mg, 48 μmol) is dissolved in 3 ml of dry pyridine and evaporated to dryness under reduced pressure. Repeat this operation three times. To this is added 5 ml of 4% tetrazole / acetonitrile solution and fluoredite and allowed to stir at room temperature. The reaction is monitored by thin layer chromatography on silica gel (TLC, developing solvent: chloroform: methanol = 9: 1). The reaction is usually complete in 2 hours. After the reaction, the solvent was driven off under reduced pressure, and 2 ml of a solution of 0.1 M iodine dissolved in tetrahydrofuran / pyridine / water = 80: 40: 2 was added thereto, and the phosphite-triester produced was stirred while stirring at room temperature. Oxidize. After 1.5 hours, the solvent is removed under reduced pressure and the remainder is extracted with chloroform. The extract is dried in the presence of anhydrous magnesium sulfate, and then the solvent is removed under reduced pressure. This is subjected to silica gel column chromatography and eluted with chloroform / methanol = 90: 10. Fluorpur with a protecting group is eluted at silica gel TLC (developing solvent: chloroform: methanol = 9: 1) at Rf 0.26. Next, the protecting group is deprotected. Fluorpur with a protecting group is added to 1 ml of a mixed solution of concentrated aqueous ammonia / ethanol = 2: 1, and β-cyanoethyl group is removed to obtain 7 mg of Fluorpur (II in FIG. 1). The synthetic product is Fluorpur because the UV-visible absorption spectrum of its pH 9 solution appears at 272 nm (from the puromycin moiety) and 494 nm (from the fluorescein moiety), and MALDI / TOF mass spectrometry [M The molecular ion of + H] + was identified by appearing at m / z 1010.
[0022]
<3> Fluorescent labeling of proteins The mRNAs produced were (1) Rabbit Reticulocyte Lysate Systems, Nuclease Treated (Promega) and (2) Wheat Germ Cell Translation System Wheat Germ Extract (Promega). Add the final concentration of Fluorpur to 16 μM in the two translation systems, and react for 60 minutes at the optimal reaction temperature (rabbit reticulocyte cell-free translation system: 30 ° C., wheat germ cell-free translation system: 25 ° C.). I let you.
[0023]
<4> Confirmation of protein labeling Confirmation of the protein labeled with fluorescylpuromycin (Fluorpur) is performed using a cell-free translation product as a sample by SDS-PAGE and a gel run for 90 minutes at 20 V constant voltage. This was performed by directly reading with a fluorescence imaging apparatus (FluorImager 595, Molecular Dynamics). The most efficient fluorescence-labeled full-length β-lactamase (molecular weight 32 kDa) uses wheat germ cell-free translation system and mRNA transcribed from DNA without a stop codon (A in Fig. 2). It was obtained when added to 16 μM (lane 1 in FIG. 3A). However, using the same wheat germ cell-free translation system, when mRNA transcribed from DNA with a stop codon (B in Fig. 2) is used even if Fluorpur has a final concentration of 16 μM, the fluorescence labeling of The efficiency was less than 1/10 compared to DNA without a stop codon (lane 3 in FIG. 3B). In addition, in the cell-free translation system for rabbit reticulocytes, the mRNA transcribed from DNA without a stop codon (lane 5 in FIG. 3A) was used for the mRNA transcribed from DNA with a stop codon. Compared to the case (lane 6 in FIG. 3A), the labeling efficiency increased 3 to 4 times. For comparison, FIG. 3B shows the same gel stained with a conventional protein fluorescent staining method (SYPRO Orange). When stained with SYPRO Orange, the protein (β-lactamase) translated by the input mRNA was buried in the protein derived from the cell-free translation system and could not be confirmed. From the above, it was confirmed that the labeling of the protein by the method of the present invention is effective in the identification of the synthetic protein by SDS-PAGE.
[0024]
【The invention's effect】
According to the present invention, it becomes possible to efficiently label the C-terminal of a protein synthesized and translated using a cell-free translation system and a living cell regardless of whether it is a prokaryotic cell or a eukaryotic cell. The present invention makes it possible to more quickly, safely and economically perform functional analysis such as identification of proteins expressed from genes and their interaction. In addition, the C-terminal labeled protein obtained by the method of the present invention is useful for identifying a compound that binds to or interacts with the protein and inhibits or activates its activity. That is, the C-terminal labeled protein obtained by the method of the present invention is detected by a method such as detecting the C-terminal label of the protein that remains or disappears as a result of binding or interaction with the compound to be screened. Compounds can also be identified.
[Sequence Listing]

[Sequence Listing Free Text]
SEQ ID NOs: 1-6: Synthetic DNA
[Brief description of the drawings]
FIG. 1 Chemical structure of puromycin and its derivatives. I is puromycin and II is fluorescyl puromycin (Fluorpur).
FIG. 2 is a construction diagram of gene DNA of β-lactamase. A is a gene DNA without a stop codon, and B is a gene DNA with a stop codon.
FIG. 3 is a polyacrylamide gel electrophoresis photograph (A) showing the binding of Fluorpur to the C-terminus of β-lactamase and a polyacrylamide gel electrophoresis photograph (B) of which it is fluorescently stained with SYPRO Orange.

Claims (4)

Composed of a label portion made of a labeling substance has a puromycin or 3 '-N-amino acyl puromycin aminonucleoside of chemical structure, the acceptor portion made of a compound having the ability to bind to the C-terminus of the translated protein In the presence of a labeling reagent, protein synthesis can be performed in a cell-free translation system or in living cells using a product transcribed from DNA consisting of a coding region with the termination codon deleted under the control of the promoter region. A method for producing a protein labeled with a C-terminus. The method according to claim 1, wherein the coding region is DNA consisting of a length corresponding to 50-3,000 amino acid residues. The method according to claim 1 or 2 , wherein the label part of the labeling reagent is a fluorescent substance, a radioactive substance, or a non-radioactive labeling substance. The method according to any one of claims 1 to 3, wherein the cell-free translation system uses wheat germ extract or rabbit reticulocyte extract.

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