JP2008193911A - Method for specifically modifying n-terminal of protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a tRNA for modifying the N-terminal of a protein on the synthesis of the protein, and to provide a method for modifying the N-terminal of the protein on the synthesis of the protein. <P>SOLUTION: An initiator tRNA is added with a carboxylic acid not having an amino group, an amide bond and a hydroxy group at the α-position. A tRNA is added with an α-hydroxy acid having a hydroxy group at the α-position and modified with at least one labeling substance. A method for producing a protein to whose N-terminal the carboxylic acid or the α-hydroxy acid is introduced with these tRNAs is also provided. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for modifying the N-terminus of a protein with a labeling substance such as a fluorescent substance, and more specifically, tRNA for modifying the N-terminus of a protein with a specific modifying substance, and the N-terminus of a protein using the tRNA. The present invention relates to a method of modifying with a specific modifying substance.

  In general, the amino group at the N-terminal of a protein is labeled with a labeling substance such as a fluorescent substance by chemical modification. However, in the method by chemical modification, since the reaction also occurs in the amino group of the lysine side chain in the protein, N-terminal specific modification is difficult.

  Protein synthesis begins with the initiation tRNA binding to the ribosome, and the α-amino acid or acylated α-amino acid added to the initiation tRNA becomes the N-terminal amino acid of the protein. In in vivo protein synthesis, methionine, valine, or their formylated form is used as the α-amino acid or acylated α-amino acid that becomes the N-terminal amino acid.

  On the other hand, it is known that unnatural amino acids other than the 20 natural amino acids and acylated amino acids in which a fluorescently labeled molecule is bonded to an α-amino group can be artificially introduced. For example, as a method for introducing an unnatural amino acid into a protein, the codon at the site to be introduced is replaced with a 4-base codon or a stop codon UAG, and the sequence corresponding to the 4-base codon or CUA is included in the anti-codon as a non-natural amino acid. There is a method of translation in the presence of aminoacylated tRNA (see Non-Patent Documents 1 to 4). By introducing a non-natural α-amino acid into a protein by such a method, functional modification or structural function analysis of the protein becomes possible.

  In addition, a method has been reported in which an amino acid labeled with a labeling substance is assigned to the start codon and an amino acid labeled with a labeling substance is introduced into the N-terminus of the protein (see Non-Patent Document 5).

  However, the molecules introduced in the conventional method for artificially introducing a specific amino acid into the N-terminus of a protein are limited to α-amino acids, acylated α-amino acids and α-hydroxy acids (see Non-Patent Document 6). It was done. This is because only a carboxylic acid having an amino group, an amide bond or a hydroxyl group at the α-position could be introduced as a molecule to be introduced by the initiation tRNA. For this reason, there was a limitation on the molecular structure of the carboxylic acid introduced into the protein N-terminus. In addition, when an amino acid is artificially introduced into the N-terminus of a protein, an artificially prepared aminoacyl tRNA is used. However, the presence of the amino group at the α-position of the aminoacyl tRNA makes the tRNA molecule unstable. For this reason, there was a problem that the tRNA was decomposed and the fluorescently labeled molecule bonded to the amino group was detached.

  On the other hand, in in vivo protein synthesis, the start codon is AUG or GUG, but this is replaced with the stop codon UAG, and the anticodon of the start tRNA is replaced with the corresponding CUA. And methods for introducing labeled amino acids have been reported (see Non-Patent Documents 7 and 8).

  Furthermore, there has also been a report on a method of assigning a hydroxy acid to a stop codon and introducing a hydroxy acid into a protein instead of an amino acid during protein synthesis (see Non-Patent Document 9). However, there has been no report on introducing a labeled hydroxy acid by using a hydroxy acid labeled with a labeling substance.

Science, 244, p.182.1989 Nucleic Acids Res., 18, 83-88, 1989 Chem. Biol., 3, 1033-1038, 1996 J. Am. Chem. Soc., 111, p. 8013, 1989 Gite S. et al., Anal. Biochem. 2000, 279, 218-225 Goto Y. et al., Nucleic Acids Symposium Series, 2006, 50, 293-294 Mamaev S. et al, Anal. Biochem. 2004, 326, 25-32 Varshney U. et al, Proc. Natl. Acad. Sci. USA. 1990, 87, 1586-1590 JA Ellman et al, Science, 1992, 255, 197-200

  An object of the present invention is to provide a tRNA for modifying the N-terminus of a protein during protein synthesis and a method for modifying the N-terminus of a protein during protein synthesis.

  The present inventors have conducted various studies on the introduction of an unnatural amino acid using a 4-base codon, but have found that an unnatural amino acid having a large fluorescent group can be introduced into a protein only with very low efficiency. . On the other hand, when these non-natural amino acids were bound to the start tRNA and introduced into the protein N-terminal by the start codon AUG, even if the protein was a large non-natural amino acid that was hardly introduced into the protein, protein N It was found that it can be introduced at the end. From these results, it was predicted that various types of unnatural amino acids could be introduced in the translation initiation process compared to the translation elongation process.

  Therefore, α-amino acid with amino group or amide bond at α-position (ie, methionine or formylmethionine) is the substrate at the start of translation, but it has no amino group, amide bond and hydroxyl group at α-position. I thought that carboxylic acid could be a substrate. When this was actually verified, it was found that a carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position is certainly a substrate at the start of translation.

  The inventors of the present invention have also conducted intensive studies on a method for modifying the N-terminus of a protein when synthesizing a protein by using a carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position. The present inventors added a carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position to tRNA. It has been newly found that when starting tRNA is used as tRNA, the above carboxylic acid can be introduced into the protein N-terminus during protein synthesis using AUG and UAG as start codons. Furthermore, it has been found that a protein in which the carboxylic acid is introduced at the N-terminus can be obtained by using a 4-base codon or other codon as a start codon instead of the start codon of AUG.

  The present inventors have found that carboxylic acids having various molecular skeletons can be introduced, and in particular, fluorescently labeled molecules using aminocarboxylic acids as spacers can be efficiently introduced into the N-terminus of proteins. Further, a protein in which a labeled α-hydroxy acid is specifically introduced at the N-terminus is obtained by binding a labeled molecule to the side chain of α-hydroxy acid, introducing the protein into a protein, and then hydrolyzing the ester bond. Has been found, and the present invention has been completed.

That is, the present invention is as follows.
[1] An initiating tRNA to which a carboxylic acid having no amino group, amide bond and hydroxyl group is added at the α-position.
[2] The starting tRNA of [1], wherein a carboxylic acid having no amino group, amide bond and hydroxyl group is added to the α-position and β-position.
[3] Carboxylic acid having no amino group, amide bond and hydroxyl group at α-position or carboxylic acid having no amino group, amide bond and hydroxyl group at α-position and β-position is modified with at least one labeling substance , [1] or [2] start tRNA.
[4] Carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position or carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position and β-position is modified with at least two labeling substances The starting tRNA of [3].
[5] The starting tRNA according to [3] or [4], wherein the modification is labeling with a fluorescent substance, a quencher substance, a cross-linking substance, digoxigenin, dinitrophenyl, cholesterol, desthiobiotin or biotin.
[6] The starting tRNA according to [1], wherein acetic acid, pyroglutamic acid, palmitoyl acid or myristoyl acid is added as a carboxylic acid.
[7] The start tRNA of any one of [1] to [6] that recognizes AUG as a start codon.
[8] The start tRNA of any one of [1] to [6] that recognizes UAG, UAA or UGA as a start codon.
[9] The start tRNA according to any one of [1] to [6], which recognizes a three-base codon other than AUG, UAG, UAA and UGA as a start codon.
[10] The start tRNA according to any one of [1] to [6], which recognizes a 4-base codon as a start codon.

[11] A carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position, or an amino group, amide bond and hydroxyl group at the α-position and β-position using the starting tRNA of any one of [1] to [10] A method for producing a protein in which a carboxylic acid having no group is specifically introduced into the N-terminus of a protein and a carboxylic acid is introduced into the N-terminus.
[12] Using an initiation tRNA modified with a labeling substance of any one of [3] to [10], a carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position, or α-position and β-position By specifically introducing a carboxylic acid having no amino group, amide bond and hydroxyl group and labeled with a labeling substance into the protein N-terminal, a protein in which the N-terminal is modified with the labeling substance is produced. Method.
[13] DNA or mRNA encoding a protein, and a four-base codon corresponding to the N-terminal amino acid of the protein, a three-base codon other than AUG, UAG, UAA, UGA, or AUG, UAG, UAA and UGA Using DNA or mRNA containing as a start codon as a template, and further using the start tRNA of any one of [1] to [10] having a sequence corresponding to the start codon of the DNA or mRNA at the anticodon site , Protein biosynthesis, specific carboxylic acid without amino group, amide bond and hydroxyl group at α-position or carboxylic acid without amino group, amide bond and hydroxyl group at α-position and β-position to protein N-terminus A method for producing a protein having a carboxylic acid introduced at the N-terminus.
[14] DNA or mRNA encoding a protein, and a 4-base codon corresponding to the N-terminal amino acid of the protein, AUG, UAG, UAA, UGA, or 3-base codon other than AUG, UAG, UAA and UGA A sequence corresponding to the start codon of the DNA or mRNA at the anticodon site, wherein the start tRNA is modified with a labeling substance of any one of [3] to [10] using DNA or mRNA containing the start codon as a template Carry out protein biosynthesis using an initiator tRNA having an amino group, an amide bond and a hydroxyl group at the α and β positions. A method for producing a protein in which the N-terminus is modified with a labeling substance by specifically introducing a carboxylic acid having no group into the N-terminus of the protein.
[15] Carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position and amino group, amide bond and hydroxyl group at the α-position and β-position produced by the method of any one of [11] to [14] A protein having a carboxylic acid having no carboxylic acid at the N-terminus, wherein the carboxylic acid is labeled with a labeling substance or not labeled.

[16] A tRNA added with an α-hydroxy acid having a hydroxyl group at the α-position and modified with at least one labeling substance.
[17] The tRNA added with the α-hydroxy acid of [16], which is modified with at least two labeling substances.
[18] tRNA to which α-hydroxy acid of [16] or [17] is added, wherein the modification is a fluorescent substance, a quencher substance, a cross-linking substance, digoxigenin, dinitrophenyl, cholesterol, desthiobiotin, or biotin .
[19] The tRNA according to any one of [16] to [18], which recognizes a 4-base codon, a stop codon, an artificial codon, or a codon assigned with an amino acid in nature.
[20] Using a fusion DNA or mRNA encoding the first protein and the second protein or peptide as a template, and further adding an α-hydroxy acid modified with any of the labeling substances of [16] to [19] A fusion protein in which the N-terminus of the first protein and the C-terminus of the second protein or peptide are bound to each other using the tRNA thus prepared, which is the N-terminal site of the first protein and the second protein or A fusion protein in which a hydroxy acid modified with a labeling substance is introduced next to the C-terminal of the peptide is prepared, and the N-terminal hydroxy acid of the first protein and the C-terminal amino acid of the second protein or peptide Comprising isolating a first protein having a hydroxy acid modified with a labeling substance introduced at the N-terminus, wherein the N-terminus is labeled In a method of manufacturing a labeled protein.
[21] A method for producing a protein wherein the second protein or peptide is a tag peptide and the N-terminus of [20] is labeled with a labeling substance.

  By using an initiation tRNA to which a carboxylic acid having no amino group, amide bond and hydroxyl group is added at the α-position, a fluorescently labeled carboxylic acid or a biotin-labeled carboxylic acid can be specifically introduced into the protein N-terminus. In many cases, the N-terminal of the protein is not related to the activity, so that fluorescent labeling or biotin labeling is possible while maintaining the protein activity. Since it is difficult to label the N-terminus specifically by the usual labeling method by chemical modification, the protein can be effectively labeled with the N-terminus according to the present invention. It is also possible to synthesize a modified protein into which acetic acid, pyroglutamic acid, myristoyl acid, or the like, which is a carboxylic acid added to the N-terminal of the protein by post-translational modification, is introduced without post-translational modification. Thereby, the structure function analysis of the N-terminal modified protein can be easily performed.

For example, by adding TAMRA-aminopropionic acid (TAMRA-AC ₃ ) to tRNA recognizing AUG as a start codon and adding streptavidin gene DNA to the E. coli cell-free translation system, TAMRA-AC ₃ is added to N Streptavidin introduced at the terminal can be synthesized.

  Another effect is that long-term protein synthesis is achieved by linking the carboxylic acid that does not have an amino group, amide bond, or hydroxyl group to the initiating tRNA, making it difficult for the ester bond between the carboxylic acid and tRNA to be degraded. Is possible. This is because when an amino group or amide bond is present at the α-position, the ester bond is likely to be decomposed under the influence of the amino group or amide bond, but the carboxyl group having no amino group or amide bond at the α-position. This is because the ester bond is stable because it is not affected by acid.

  On the other hand, α-hydroxy acids are known to be introduced into proteins in the translation system in the same manner as α-amino acids. However, since the resulting ester bond is more susceptible to hydrolysis than an amide bond (peptide bond), a protein cleaved immediately before the α-hydroxy acid is generated. As a result, a protein having an α-hydroxy acid at the N-terminus of the protein is obtained. Therefore, by attaching a labeling molecule such as a fluorescent label or biotin to the side chain of α-hydroxy acid, a protein having the N-terminal can be synthesized.

Hereinafter, the present invention will be described in detail.
Carboxylic acids having no amino group, amide bond and hydroxyl group at the α-position are, for example, those represented by the general formula (I)

COOH-X-Y-R ₁ (I)

It is represented by

In general formula (I), X is a divalent group, preferably —CH ₂ —. C contained in X is α carbon. Carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position means a carboxylic acid in which no amino group and hydroxyl group are bonded to the α carbon, and the α carbon is not bonded to other groups by an amide bond. Say. X is not —C (NH ₂ ) — or —C—NH— or —C (OH) —.

The present invention further includes carboxylic acids having no amino group, amide bond and hydroxyl group at the α-position and β-position as well as the α-position. Carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position and β-position, when the above general formula (I) is represented by COOH—X—X′—Y ′ (X contains one carbon) A divalent group, preferably —CH ₂ —), and C contained in X ′ is a β-carbon. Carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position and β-position does not have an amino group and a hydroxyl group bonded to the α-carbon and β-carbon, and the α-carbon and β-carbon are bound by other amide bonds. A carboxylic acid that is not bound to a group. X ′ is not —C (NH ₂ ) — or —C—NH— or —C (OH) —, and X ′ is preferably —CH ₂ —. In the present invention, when it is referred to as a carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position, it may contain a carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position and β-position.

  Y in the general formula (I) functions as a spacer between the carboxylic acid and the labeling substance when the carboxylic acid is modified with the labeling substance. The term “spacer” refers to a portion connecting a carboxylic acid moiety and a labeling substance in a labeled carboxylic acid molecule to which a labeling substance is bound. That is, when the side chain in the labeled carboxylic acid molecule is not directly bonded to the labeling substance, but one or more atoms are present between the side chain and the labeling substance, the carboxylic acid of the labeled carboxylic acid It is said that the acid part and the labeling substance are linked via a spacer. The spacer represented by Y in the general formula (I) may contain at least one of C, O, N and S in the main chain portion. However, atoms adjacent to X are other than N and O (when N or O are adjacent, these correspond to α-amino acid and α-hydroxy acid derivatives, respectively). The main chain structure of the spacer is preferably one in which the above atoms are 2 to 30, preferably 2 to 20, more preferably 2 to 10, more preferably 3 to 8, and linearly bonded. One or more double bonds may be contained in the chain structure. Furthermore, the spacer may have 1 to several, preferably 1 to 5, more preferably 1 to 3, cyclic structures such as a benzene ring and / or a cyclohexyl ring. Moreover, the thing of the structure where cyclic structures, such as a benzene ring and a cyclohexyl ring, or the cyclic structure and the said linear structure were combined may be sufficient. For example, Y is a divalent group derived from an aromatic hydrocarbon ring (aryl), alkane, alkene or alkyne.

  Furthermore, the spacer part may have a side chain, and another labeling substance may be bound to the side chain.

  Depending on the type of labeling substance, in order to more effectively exert its function in the introduced protein, it may be advantageous to bind to a carboxylic acid via a spacer. For example, depending on the type of the labeling substance, it can be considered that steric hindrance to the labeling compound in the introduced protein is reduced by binding via a spacer.

Specific examples of Y include, for example, —CH ₂ —, — (CH ₂ ) m — (m is 1 to 7), — (CH ₂ ) o —NH—CO— (CH ₂ ) p — (o is 1 to 7, p is _{1~5), - O- (CH 2} ) 2 -O- (CH 2) 2 -, - (CH 2) qC 6 H 4 - (q is 1-3), and include However, it is not limited to these.

  In addition, when general formula (I) is represented by COOH-X-X'-Y ', Y' is represented by a chemical formula obtained by removing a divalent group represented by X 'from Y.

R ₁ is a functional group such as an amino group, a thiol group, a carboxyl group, a hydroxyl group, an aldehyde group, an allyl group, or a halogenated alkyl group, and is preferably an amino group.

When R ₁ is an amino group, it may be called an aminocarboxylic acid. When Y is CH ₂ and R ₁ is an amino group, it may be referred to as aminopropionic acid.

The α-amino acid modified with two labeling substances is, for example, the general formula (II),

COOH-CH (NR ₂ ) -Y-R ₁ (II)

It is represented by

In the general formula (II), NR ₂ represents a labeled amino group. For example, NR ₂ has a structure in which a labeling substance is bonded to an amino group using a compound such as succinimide ester, isothiocyanate, sulfonyl chloride, NBD-halide, dichlorotriazine. The labeling substance to be bound is not limited, and examples thereof include biotin. A spacer having a main chain structure containing at least one of C, O, N, and S may be included between biotin and carboxylic acid. For example, a group corresponding to the structure of lysine may be included.
Formula (II) Y and R ₁ in are respectively the same as Y and R ₁ in formula (I).

Further, the α-hydroxy acid having a hydroxyl group at the α-position is, for example, the general formula (III)

COOH-CH (OH) -Z-R ₁ (III)

It is represented by

In general formula (III), Z is a part corresponding to a side chain of an α-amino acid. In view of being easily incorporated into ribosomes, —CH ₂ —C ₆ H ₄ — is preferable.
R ₁ in the general formula (III) is the same as R ₁ in formula (I).

In the present invention, the α-hydroxy acid having a hydroxyl group at the α-position represented by the general formula (III) is labeled with at least one labeling substance, and the labeling substance is bonded via, for example, R ₁ . The α-hydroxy acid having a hydroxyl group at the α-position of the present invention does not have an amino group or an amide bond at the α-position.

  To add a carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position or a hydroxy acid having a hydroxyl group at the α-position to tRNA, dinucleotide (pdCpA) is bound to the carboxyl group of the carboxylic acid or hydroxy acid. The tRNA (tRNA (-CA)) lacking the CA dinucleotide at the 3 'end can be combined with a ligase such as T4 ligase. Artificial tRNA can be produced according to the description of the method for producing tRNA containing an artificial amino acid described in International Publication No. WO2004 / 009709 pamphlet and the like.

  The tRNA to be used is not limited. Examples of the starting tRNA for introducing a carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position include Escherichia coli, yeast, Mycoplasma capricolum, and Bacillus halodolance. Initiating tRNAs such as halodouranns) and Mycoplasma pneumoniae can be used. Further, tRNA for introducing α-hydroxy acid includes tRNA for yeast phenylalanine (Science, 244, p.182.1989; Nucleic Acids Res., 18, 83-88, 1989), tRNA for E. coli asparagine, detrahimenaglutamine. TRNA (Chem. Biol., 3, 1033-1038, 1996), tRNA for E. coli glycine (J. Am. Chem. Soc., 111, p. 8013, 1989) and the like can be used. Also, E. coli, Mycoplasma capricolum, Bacillus halodourans, Bacillus subtillis, Borreiia burgdorferi, Mycoplasma genitalium, Mycoplasma genitalium, Mycoplasma genitalium TRNA corresponding to tryptophan derived from microorganisms such as (Mycoplasma pneumoniae) and Staphylococcus aureus N315 can also be used.

  These tRNA anticodons may be modified to those corresponding to the codons used.

  The tRNA having an amino group, an amide bond and a carboxylic acid having no hydroxyl group added at the α-position and the tRNA having an α-hydroxy acid having a hydroxyl group added at the α-position are preferably at least one or two labels. It is modified with a substance.

  The labeling substances are dye compounds, fluorescent substances, chemi / bioluminescent substances, enzyme substrates, coenzymes, antigenic substances and protein binding substances known to those skilled in the art. Further examples include quencher substances, cross-linking substances, digoxigenin, dinitrophenol, cholesterol, desthiobiotin, biotin and the like. Here, the quencher substance (quenching substance) refers to a substance that suppresses the fluorescence of the fluorescent substance when it is present in the vicinity of the fluorescent substance. Specific examples include dinitrobenzene, dimethylaminoazobenzene, Cy5Q. , Cy7Q. In addition, the cross-linking substance refers to a substance that can form a covalent bond with an adjacent substance by light irradiation or the like, and specific examples include benzophenone, psoralen, and trifluoromethylphenyl diazirine.

  In addition, usually, post-translational modification adds a carboxylic acid such as acetic acid, pyroglutamic acid, palmitoyl acid or myristoyl acid to the N-terminus of the protein, so that the protein exhibits its normal function. The starting tRNA added with a carboxylic acid of the present invention also includes an starting tRNA added with a carboxylic acid such as acetic acid, pyroglutamic acid, palmitoyl acid, myristoyl acid. By using these initiation tRNAs, carboxylic acids such as acetic acid, pyroglutamic acid, palmitoyl acid, myristoyl acid can be introduced into the N-terminal during protein translational synthesis.

  Examples of the fluorescent substance that can be used in the present invention include fluorescent substances having Rhodamine, Cy, Coumarine, and EvoBlue as a basic skeleton. Specifically suitable as a fluorescent substance to be bound to the carboxylic acid of the present invention, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, EVOblue10, EVOblue30, SFX, RhoX, TexX, TAMX, 5TAM, FAM, etc. Can be mentioned. Furthermore, derivatives of these fluorescent substances are also included. Further, fluorescent substances having naphthalene, biphenyl, anthracene, phenenthrene, pyrene, carbazole or derivatives thereof are also included.

In addition, other fluorescent materials, specifically, BFL, BFLC5, B493, BR6G, B558, B564, B576, B581, and BFL (trade names and / or general names are described below) can also be used.

BFL: BODIPY FL (trade name),
4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indancene-3-propionic acid

BFLC5: BODIPY FL C5 (trade name),
4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s- indancene-3-pentanoic acid

B493: BODIPY 493/503 (trade name),
4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a, 4a-diaza-s-indancene-8-propionic acid

BR6G: BODIPY R6G (trade name),
4,4-difluoro-5- (4-phenyl-1,3-butadienyl) -4-bora-3a, 4a-diaza-s-indancene-3-propionic acid

B558: BODIPY 558/568 (trade name),
4,4-difluoro-5- (2-thienyl) -4-bora-3a, 4a-diaza-s-indancene-3-propionic acid

B564: BODIPY 564/570 (trade name),
4,4-difluoro-5-styryl-4-bora-3a, 4a-diaza-s-indancene-3-propionic acid

B576: BODIPY 576/589 (trade name),
4,4-difluoro-5- (2-pyrrolyl) -4-bora-3a, 4a-diaza-s-indancene-3-propionic acid

B581: BODIPY 581/591 (trade name),
4,4-difluoro-5- (4-phenyl-1,3-butadienyl) -4-bora-3a, 4a-diaza-s-indancene-3-propionic acid

Cy3 (trade name), Cy3B (trade name), Cy3.5 (trade name), Cy5 (trade name), Cy5.5 (trade name), Cy5Q (trade name), Cy7Q (trade name), EvoBlue 10 (trade name) ), EvoBlue30 (trade name)

SFX: SFX (trade name) is a trade name when succinimide ester is bonded, and FAM-X is a trade name without ester.
RhoX: Rhodamine Red-X (trade name)
TexX: Texas Red-X (trade name)
TAMX: 5 (6) -TAMRA-X (trade name)
5TAM: 5 TAMRA (trade name)

  As the fluorescent substance used as the labeling substance, those having an excitation wavelength in the visible light range (near 400 to 700 nm) and further having an emission wavelength in the visible light range are preferred, and those having a strong emission intensity in an aqueous solution are particularly preferred. .

  A chemical or bioluminescent substance such as luciferin or Lumigen or a derivative thereof can also be used as a labeling substance in the present invention.

  Furthermore, coenzymes, substances with antigenicity, and substances known to bind to specific proteins are selected as appropriate according to the function to be given to the target protein, and this is used as a labeling substance. Can be used for invention. For example, if a substrate for a specific enzyme (for example, a substrate for alkaline phosphatase or β-galactosidase) is introduced, it can be detected using a color reaction by the enzyme. Proteins labeled with an antigenic substance or a substance that is known to bind to a specific protein can be used for indirect detection methods using antibodies or binding proteins, and can be easily purified. It has the advantage of being. For example, a functional protein whose N-terminal is modified with biotin using the method of the present invention has a function of binding to avidin or streptavidin via biotin, and if this function is used, It is possible to establish a detection system for a specific substance by a method or using a bond with avidin or streptavidin chemically labeled with a fluorescent substance or the like. In addition, those skilled in the art can understand that various dyes and substances detectable by various biochemical, chemical, or immunochemical detection methods can be used as the labeling substance.

The bond between the carboxylic acid or the hydroxy acid and the labeling substance can be achieved by using the bond between the functional group of the R ₁ moiety of the above general formula (I) and the labeling substance functional group bound to the carboxylic acid or hydroxy acid of the present invention. Good. That is, as the R ₁ portion of the spacer bonded to the carboxylic acid represented by the general formula (I) or the hydroxy acid represented by the general formula (III), an amino group that does not participate in the peptide elongation reaction during protein synthesis, A functional group such as a thiol group, a carboxyl group, a hydroxyl group, an aldehyde group, an allyl group, or an alkyl halide group is introduced, and a labeling substance can be bonded to the functional group via a spacer. If R ₁ moiety is an amino group, succinimide ester as the amino group-labeled probe, isothiocyanate, sulfonyl chloride, NBD-halide compound, such as dichlorotriazine is when R ₁ moiety is a thiol group, a thiol group for labeling probes When a compound such as an alkyl halide, maleimide, or aziridine has a carboxyl group as the R ₁ moiety, a diazomethane compound, an aliphatic bromide, a carbodiimide, or the like can be used as a probe for labeling a carboxyl group. For example, a succinimide ester can be introduced into a labeling substance via a spacer, an amino group can be introduced into an aromatic ring in the spacer bonded to a carboxylic acid, and both can be bound by an amide bond. For a method of bonding functional groups to each other, for example, the description of “Shinsei Kagaku Kogaku Kenkyu 1 Protein VI Structure-Function Relationship” may be referred to.

  When preparing a tRNA to which a carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position or a hydroxy acid having a hydroxyl group at the α-position and further modified with a labeling substance is prepared, After modifying acid or hydroxy acid with a labeling substance, pdCpA may be bound and added to tRNA, or after adding carboxylic acid or hydroxy acid to tRNA or pdCpA, the carboxylic acid or hydroxy acid part is labeled with the labeling substance May be.

In the present invention, a compound represented by COOH- (CH ₂ ) _n-1 -NH-F (F is a labeling substance, n is a natural number) is represented by F-AC _n and COOH- (CH ₂ ) _n-1 -NH- A compound represented by CO— (CH ₂ ) ₅ —NH—F (F and n are as defined above) may be referred to as FX-AC _n . In addition, a compound represented by COOH- (CH ₂ ) _n-1 -B-NH-F (B is an aromatic ring, F and n are the same as above) is represented by F-ABC _n , COOH-CH ₂ -O- ( A compound represented by CH ₂ ) ₂ —O— (CH ₂ ) ₂ —NH—F (F is the same as above) may be referred to as F-TEG. Further, COOH—CH (OH) —CH ₂ —C ₆ H ₄ —NH—F (F is the same as above) having a hydroxyl group at the α-position may be referred to as F-AFL. COOH—CH (OH) —CH ₂ —C ₆ H ₄ —NH—CO— (CH ₂ ) ₅ —NH—F (F is the same as above) may be referred to as FX-AFL.

  1A to 1D show carboxylic acids having no amino group, amide bond and hydroxyl group at the α-position, to which TAMRA is bound as a labeling substance. 1E and F are those having an amino group at the α-position to be compared (FIG. 1E) and those having an amide bond (FIG. 1F).

  2A to 2E show carboxylic acids having BODIPY FL bound as a labeling substance and having no amino group, amide bond and hydroxyl group at the α-position. 2F and G are those having an amino group (FIG. 2F) at the α-position to be compared and those having an amide bond (FIG. 2G).

  3A to 3E show carboxylic acids having no amino group, amide bond and hydroxyl group at the α-position, to which various fluorescent substances (BODIPY FL, FAM, TAMRA, Cy3 and Cy5) are bound as labeling substances.

  FIG. 4A shows α-amino acids labeled with two labeling substances TAMRA and biotin. FIG. 4B shows a carboxylic acid that does not have an amino group, an amide bond and a hydroxyl group at the α-position and is labeled with two substances, TAMRA and biotin.

  5B and 5D show hydroxy acids in which BODIPY FL or TAMRA is bound as a labeling substance at the α-position. 5A and 5C are α-amino acids having an amino group at the α-position to be compared.

For example, in the compound having the structure represented by FIGS. 1A to 1E, COOH—CH ₂ —Y—NH—CO—F (Y is the same as Y in the compound represented by the above general formula (I), and F is In the formula (I), R ₁ is NH ₂ and COOH—CH ₂ —Y—NH ₂ represents an amino group at the α-position, It corresponds to a carboxylic acid having no amide bond and hydroxyl group.

FIG. 6 shows an example of a carboxylic acid structure (BODIPY FL-ABC ₃ -pdCpA) bound to pdCpA.
The introduction of the carboxylic acid of the present invention into the N-terminus of the protein can be carried out by assigning a carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position of the present invention to the initiation codon. That is, the anticodon of tRNA to which a carboxylic acid having no amino group, amide bond and hydroxyl group is added at the α-position may be made to correspond to the start codon. The initiation codon refers to a codon that is the starting point for protein synthesis when mRNA is translated into protein. In general, AUG is the start codon, but in the present invention, it is not limited to AUG, and any codon of 4-base codon, stop codon, artificial codon and other 3-base codons is used for mRNA used to synthesize the target protein. Can be used as an initiation codon. In this case, the tRNA can be arbitrarily selected by making the anticodon part of the tRNA added with a carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position complementary to the codon used as the start codon. Is recognized as the start codon. The tRNA that recognizes the start codon and acts first in the protein synthesis process is called the start tRNA. For example, any codon at the 5 ′ end of the region encoding the protein in mRNA can be the start codon. When the initiation codon is assigned to the carboxylic acid of the present invention, a carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position of the present invention is introduced at the N-terminus of the synthesized protein. At this time, the N-terminal of the protein is modified with the labeling substance by using a carboxylic acid modified with the labeling substance.

  As described above, the codon can be extended by assigning a carboxylic acid to a codon that is not normally assigned an amino acid, specifically, a 4-base codon, a stop codon, or an artificial codon. Four base codons are described in Hohsaka T., et al., J. Am. Chem. Soc., 118, 9778-9779, 1996 and Hohsaka T., et al., J. Am. Chem. Soc., 121, 34. -40, 1999 can be used as a reference. Stop codons are Science, 244, p.182.1989 and J.Am.Chem.Soc., 111, p.8013,1989, and artificial base codons are Hirao, I., et al., Nature Biotech., 20 , 177-182, 2002, can be used as a reference.

  When a 4-base codon is used, the bases constituting the 4-base codon are not limited, but examples of the 4-base codon include CGGG, CTCT (CUCU), CTCA (CUCA), CCCT (CCCU), CGGT (CGGU), AGGT (AGGU ) And GGGT (GGGU) are preferably used. Examples of stop codons include UAA, UAG, and UGA.

  When the 4-base codon method is used, a 4-base codon is incorporated into the codon site at the 5 ′ end of the DNA or mRNA encoding the target protein to be modified at the N-terminus. In addition, the anticodon site of tRNA added with a carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position of the present invention is substituted with the corresponding 4 bases. When these are used to synthesize proteins, a 4-base codon-anticodon pair is formed in the ribosome at the part substituted with a 4-base codon, and an amino group, an amide bond and a hydroxyl group are added to the α-position bound to tRNA. Synthesis of a protein in which a carboxylic acid that does not exist is present at the N-terminus is initiated. On the other hand, since the other part is translated by 3 bases as usual, a carboxylic acid is introduced only into the N-terminal part designated by the 4-base codon in the finally obtained protein. In the 4-base codon method, not only a codon consisting of 4 consecutive bases but also a codon consisting of 5 or more consecutive bases can be used. For example, a codon consisting of 5, 6 or 7 consecutive bases can be mentioned. When using a codon consisting of 5 or more consecutive bases, the anticodon site of tRNA may be replaced with the corresponding 5 or more bases. In the present invention, a 4-base codon includes a codon consisting of 4 consecutive bases changed to a codon consisting of 5 or more consecutive bases.

  In addition, codons that are assigned amino acids in nature can also be used. In this case, in the introduction of the protein, the carboxylic acid and the natural amino acid compete with each other, and the production efficiency of the protein in which the carboxylic acid is introduced at the N-terminus may be lowered. At this time, if a carboxylic acid is assigned to a rare codon that is not frequently used in nature, the production efficiency may be increased. The rare codon varies depending on the translation system to be used. For example, when using an E. coli translation system, a rare codon in E. coli may be used. Examples of rare codons of E. coli include AGG, AGA, AUA, CUA, CCC, GGA, CGG and the like.

  When using any of the four-base codon method, the stop codon method, or the artificial codon method, or when using codons to which amino acids are naturally assigned, DNA or mRNA that encodes the protein to be synthesized. Can be incorporated at the 5 'end.

  Similarly, when the tRNA to which the α-hydroxy acid of the present invention is added is used, a DNA or mRNA encoding another protein or peptide is fused to the 5 ′ end of the DNA or mRNA encoding the protein to be synthesized. A 4 base codon that is an extension codon other than the start codon, a stop codon, an artificial codon, or a codon assigned with an amino acid in nature may be incorporated into the 5 ′ end of the DNA or mRNA encoding the protein to be synthesized. . Here, the target protein to be synthesized is called a first protein, and the other protein or peptide is called a second protein. In this case, when the protein synthesis proceeds, the second protein is first translated, and after the translation is finished, the present invention corresponding to the above-mentioned 4-base codon, stop codon, artificial codon, or a codon assigned with an amino acid in nature The tRNA to which the α-hydroxy acid is added binds to the codon part. Then, since the carboxyl group of the C-terminal amino acid of the second protein and the hydroxyl group of the α-hydroxy acid are bonded by an esterification reaction, a fusion protein of the first protein and the second protein is generated. In the fusion protein, the hydroxy acid is introduced at the N-terminus of the first protein and next to the C-terminus of the second protein. In this case, by hydrolyzing the fusion protein, the ester bond between the C-terminal amino acid and the hydroxy acid of the second protein or peptide is cleaved, and the hydroxyl acid is introduced into the N-terminal. A protein, that is, a first protein in which the N-terminus is labeled with a labeling substance can be obtained. The hydrolysis treatment may be performed using an acid or base as a catalyst. Depending on the type of the second protein, hydrolysis proceeds automatically, and the first protein with the N-terminal modified, which is a free target protein, can be obtained. In the present invention, “hydrolyze” includes not only the case where the hydrolysis treatment is performed artificially but also the case where the hydrolysis occurs automatically. Here, the other protein or peptide which is the second protein is not limited, but may be a protein having a small number of amino acids. For example, a peptide generally used as a tag peptide can be used. As the tag peptide, 2 to 12, preferably 4 or more, more preferably 4 to 7, more preferably 5 or 6 histidine polyhistidine tag, T7 tag, RGSHis3 tag, VSV-G tag, Examples include Avi tags (tags composed of biotin-accepting peptides), Glu-Glu (EE) tags, FLAG tags, HA tags, and the like. Examples of the protein include glutathione-S-transferase (GST), maltose binding protein (MBP) and the like.

  When a 4-base codon assigned to the hydroxy acid of the present invention is linked to the 5 ′ end of DNA or mRNA encoding the first protein, the second protein that is the target protein downstream of the 4-base codon is used. When linking these proteins, a sequence corresponding to the stop codon in the +1 frame may be included upstream of the 4-base codon, and a sequence corresponding to the stop codon in the +2 frame may be included downstream of the 4-base codon. In addition, 2 + 3 × A bases are inserted between the sequence corresponding to the stop codon in the upstream frame of the 4-base codon and the 4-base codon, and between the sequence corresponding to the stop codon in the downstream +2 frame and the 4-base codon. It may be. By inserting such a base, protein translation stops when a frame shift occurs in the translation process of the protein, so that unnecessary protein synthesis can be avoided. The stop codon is represented by 3 bases of TAA or UAA, TAG or UAG, or TGA or UGA. In the above “2 + 3 × A”, A is 0 or a positive integer, and there is no upper limit to the number of A, and it is sufficient that the protein synthesis is finally stopped. However, from the viewpoint of quickly stopping the synthesis of a protein in which a frame shift has occurred, it should not be too large. Preferably A is 10, 9, 8, 7, 6, 5, 4, 3 or 2 or less, or 1 or 0.

  In the method of introducing a carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position or a hydroxy acid having a hydroxyl group at the α-position into the N-terminus of the protein of the present invention, a synthetic system using living cells and a cell-free system Although a protein synthesis system can be used, it is preferable to use a cell-free protein synthesis system.

  In order to obtain a protein in which a carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position or a hydroxy acid having a hydroxyl group at the α-position is introduced at the N-terminus by a protein synthesis system using living cells, The protein of the present invention can be expressed in living cells using the method of injection by microinjection (Science, 268, p.439, 1995).

  Synthesis by cell-free translation system can be performed by introducing the expression vector containing the gene to be expressed into the host cell by mixing with the necessary reagents in vitro and expressing the gene (Spirin, AS et al, (1988) “A continuous cell-free translation system capable of production performance in high yield” Science 242, 1162; Kim, DM, et al., (1996) “A highly efficient cell-free protein synthesis system from E .coli ”Eur. J. Biochem. 239, 881-886). The cell-free protein synthesis system may refer only to the cell-free translation system that reads the genetic information of mRNA and synthesizes the protein on the ribosome, or the cell-free transcription system that synthesizes RNA using DNA as a template Sometimes it includes both translation systems. In cell-free translation systems, biological extracts are used. Biological extracts are ribosomes, 20 types of aminoacyl-tRNA synthetases, methionyl-tRNA transformylase, 3 types of translation initiation factors (IF1, IF2, IF3), 3 types of translation elongation factors (translation elongation) factor: EF-G, EF-Tu, EF-Ts), three types of translation termination factors (RF1, RF2, RF3), ribosome recycling factor (RRF), required for protein synthesis such as RNA polymerase An organism extract containing ingredients. Proteins other than those listed here may be added for efficient translation, and those skilled in the art can determine what proteins should be added for more efficient addition. The biological extract may be any of E. coli-derived, wheat germ-derived, rabbit reticulocyte-derived, animal cell or insect cell-derived. The biological extract can be obtained by crushing using a French press or crushing using glass beads. The Escherichia coli-derived microbial extract includes S30 extract, which can be obtained by the method of Pratt et al. (Pratt, Transcription and Translation-a practical approach, Henes, BD and Higgins, SJ ed., IRL Press, Oxford., 179-209 [1984]). S30 extract consists of ribosome, 20 types of aminoacyl-tRNA synthetase, methionyl-tRNA transformylase, 3 types of translation initiation factor (IF1, IF2, IF3), 3 types of translation elongation factor EF-G, EF-Tu, EF-Ts), three types of translation termination factors (RF1, RF2, RF3), ribosome recycling factor (RRF) and the like. The cell-free protein synthesis system includes, in addition to the above biological extract, an ATP regeneration system, a promoter and a plasmid containing a nucleic acid encoding the protein to be expressed or mRNA, tRNA, RNA polymerase encoding the protein to be expressed, RNAase inhibitors, energy sources such as ATP, GTP, CTP, UTP, buffers, amino acids, salts, antibacterial agents and the like may be contained, and the respective concentrations may be appropriately determined.

  The ATP regeneration system is not limited, and a known combination of phosphate donor and kinase can be used. Examples of this combination include phosphoenolpyruvate (PEP) -pyruvate kinase (PK), creatine phosphate (CP) -creatine kinase (CK), acetyl phosphate (AP) -acetate kinase (AK) Combinations and the like may be mentioned, and these combinations may be added to the cell-free protein synthesis system.

  The cell-free protein synthesis system also requires mRNA encoding the protein to be produced. The mRNA may include a system that transcribes a nucleic acid in a cell-free protein synthesis system, that is, a system that produces mRNA encoding the protein. In this case, DNA is added. Alternatively, mRNA may be separately synthesized by transcription or the like, and the obtained mRNA may be included in the cell-free protein synthesis system of the present invention. The production of mRNA can be achieved by a plasmid containing a suitable promoter and DNA encoding the protein to be produced located downstream of the promoter, and RNA polymerase acting on the promoter. Here, the plasmid to be used is not limited, and a known plasmid can be used. It can be used by introducing an appropriate promoter, ribosome binding site, etc. by a known genetic engineering technique. A person skilled in the art can appropriately select a plasmid used in the present invention and design and construct it by himself. As the promoter, an endogenous promoter possessed by a microorganism used in the cell-free protein synthesis system may be used, or an exogenous promoter may be used. As the promoter, the above Trc promoter, T7 promoter and Tac promoter are excellent in terms of efficiency and are preferably used.

  Proteins can be expressed using commercially available cell-free expression kits. Examples of such kits include Rapid Translation System (RTS) (Roche) and Expressway In Vitro Protein Synthesis System (Invitrogen). In this case, the expression vector to be used is not limited, but a vector suitable for each cell-free translation system may be used. Examples of the former kit expression vector include pIVEX2.2bNde, and examples of the latter kit expression vector include pEXP1 and pEXP2.

  By using the tRNA of the present invention, a carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position or a hydroxy acid having a hydroxyl group at the α-position can be introduced at the N-terminus of the protein. Alternatively, when the hydroxy acid is modified with a labeling substance, the N-terminus of the protein can be modified with the labeling substance.

  The synthesized N-terminal modified protein can be appropriately purified according to the properties of the protein. For example, it can be purified by molecular weight by SDS polyacrylamide gel electrophoresis or gel filtration. Further, depending on the charged state, it can be purified by ion exchange chromatography. Furthermore, it can be purified by affinity chromatography using a substance that specifically binds to the protein of interest, such as a specific antibody.

  When the N-terminus of a protein is modified with a labeling substance, the activity of the protein is not easily affected by the labeling substance, so that a modified protein that retains the original activity of the protein can be obtained.

  When protein synthesis is performed, when tRNA added with an artificial amino acid such as a labeled amino acid to which a labeling substance is bound is used, the tRNA has a free α-amino group, and an ester between the tRNA and the amino acid. Bonds are susceptible to hydrolysis. The tRNA added with a carboxylic acid having no amino group, an amide bond and a hydroxyl group at the α-position or an α-hydroxy acid having a hydroxyl group at the α-position and having a labeling substance bound thereto is not easily degraded. Therefore, a protein having an N-terminal modified efficiently can be produced.

  By using the tRNA of the present invention, the N-terminus of any protein can be modified.

  The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

Example 1 Synthesis of tRNA in which carboxylic acid having no amino group, amide bond and hydroxyl group at α-position is modified with at least one labeling substance (1) Synthesis of aminocarboxylic acid-pdCpA For 100 mg of aminocarboxylic acid 2 equivalents of NaHCO _3, 2 mL of water and 2 ml of Dioxane were added, and while stirring under ice-cooling, (Boc) ₂ O 1.5 equivalents / 200 μL of Dioxane was added dropwise and reacted for 18 hours. Ethyl acetate was added, and the mixture was washed 3 times with 5% potassium hydrogen sulfate and then once with saturated brine. Thereafter, anhydrous magnesium sulfate was added for dehydration, followed by filtration and drying with a centrifugal evaporator to obtain Boc-aminocarboxylic acid.

  Acetonitrile (2 mL) and Triethylamine (500 μL) were added to 100 mg of the obtained Boc-aminocarboxylic acid, and Chloroacetonitrile (200 μL) was added dropwise with stirring under ice-cooling for 18 hours. Ethyl acetate was added, and the mixture was washed 3 times with 5% potassium hydrogen sulfate, 3 times with 4% sodium hydrogen carbonate, and once with saturated brine. Thereafter, anhydrous magnesium sulfate was added for dehydration, followed by filtration and drying by a centrifugal evaporator to obtain a cyanomethyl ester of Boc-aminocarboxylic acid.

To 15 μL (0.66 μmol) of pdCpA in DMF, 5 equivalents of Boc-aminocarboxylic acid cyanomethyl ester was added and reacted at room temperature for 3 hours (however, AC ₂ was on ice for 30 minutes, AC ₁₂ was at room temperature) 72 hours, ABC _{2 to} ABC ₄ were reacted at 37 ° C. for 3 hours). 600 μL of diethyl ether was added and stirred, and the ether was removed after centrifugation. The precipitate was washed twice with 600 μL of ether and then dried under reduced pressure. Subsequently, 200 μL of trifluoroacetic acid was added and left on ice for 15 minutes to remove Boc. After concentration under reduced pressure with a centrifugal evaporator, 600 μL of ether was added and stirred. After centrifugation, ether was removed. This operation was further repeated twice to wash and then dried under reduced pressure. A part of the obtained aminocarboxylic acid-pdCpA was hydrolyzed with 0.1M NaOH, the concentration was calculated from the peak area of the resulting pdCpA, and dissolved in DMSO to be 2.2 nmol / μL.

(2) Synthesis of TAMRA-aminocarboxylic acid-pdCpA (other than ABC _{2 to} ABC ₄ )
30 μL (66 nmol) of a DMSO solution of aminocarboxylic acid-pdCpA and 3 μL (150 nmol) of a 50 mM DMSO solution of 5-TAMRA-SE were mixed with 27 μL of DMSO. Subsequently, an equal amount of 0.1M NaHCO ₃ was added and reacted on ice for 1 h, and then neutralized by adding 9 μL of 1M AcOH. 200 μL of chloroform was added and stirred. After centrifugation, the chloroform phase was removed, and this operation was further repeated 5 times. The target peak was separated by HPLC, and the solvent was removed by a centrifugal evaporator. A part of the obtained TAMRA-aminocarboxylic acid-pdCpA was hydrolyzed with 0.1 M NaOH, the concentration was calculated from the peak area of the resulting pdCpA, and dissolved in DMSO to be 2.2 nmol / μL.

(3) Synthesis of TAMRA-aminocarboxylic acid-pdCpA (ABC _{2 to} ABC ₄ )
40 μL (88 nmol) of aminocarboxylic acid-pdCpA and 4 μL (200 nmol) of 50 mM DMSO solution of 5,6-TAMRA-X-SE were mixed. Subsequently, an equal amount of 1M Pyridine-HCl aqueous solution (pH 5.0) was added and reacted at 37 ° C. for 3 hours. 200 μL of chloroform was added and stirred. After centrifugation, the chloroform phase was removed, and this operation was further repeated 5 times. The target peak was separated by HPLC, and the solvent was removed by a centrifugal evaporator. A part of the obtained TAMRA-aminophenylcarboxylic acid-pdCpA was hydrolyzed with 0.1M NaOH, the concentration was calculated from the peak area of the resulting pdCpA, and dissolved in DMSO to be 2.2 nmol / μL. .

  1A-D show the structure of TAMRA-aminocarboxylic acid. TAMRA-aminocarboxylic acid-pdCpA is bound to pdCpA via the carboxyl group of the carboxylic acid.

(4) Synthesis of BODIPY FL-aminocarboxylic acid-pdCpA (other than ABC _{2 to} ABC ₄ )
15 μL (33 nmol) of aminocarboxylic acid-pdCpA in DMSO and 1.5 μL (150 nmol) of 100 mM DMSO in BODIPY FL-SE (use BODIPY FL-X-SE for X-AC ₁₂ ) were mixed with 13.5 μL of DMSO . Subsequently, 30 μL of 0.1M NaHCO ₃ was added and reacted on ice for 1 h, and then neutralized by adding 4.5 μL of 1M AcOH. The target peak was separated by HPLC, and the solvent was removed by a centrifugal evaporator. A part of the obtained BODIPY FL-aminocarboxylic acid-pdCpA was hydrolyzed with 0.1M NaOH, the concentration was calculated from the peak area of the resulting pdCpA, and dissolved in DMSO to be 2.2 nmol / μL. .

(5) Synthesis of BODIPY FL-aminocarboxylic acid-pdCpA (ABC _{2 to} ABC ₄ )
30 μL (66 nmol) of aminocarboxylic acid-pdCpA and 3 μL (300 nmol) of a 100 mM DMSO solution of BODIPY FL-SE were mixed. Subsequently, 33 μL of 1M Pyridine-HCl aqueous solution (pH 5.0) was added and reacted at 37 ° C. for 3 hours. The target peak was separated by HPLC, and the solvent was removed by a centrifugal evaporator. A part of the obtained BODIPY FL-aminocarboxylic acid-pdCpA was hydrolyzed with 0.1M NaOH, the concentration was calculated from the peak area of the resulting pdCpA, and dissolved in DMSO to be 2.2 nmol / μL. .

  2A to E show the structures of BODIPY-aminocarboxylic acids. BODIPY-aminocarboxylic acid-pdCpA is bound to pdCpA via the carboxyl group of the carboxylic acid.

(6) Synthesis of BODIPY FL-pdCpA
10 μL of 0.5 M acetonitrile solution of BODIPY FL and 0.5 μL of 1 M acetonitrile solution of 1,1′-Carbonyl diimidazole were mixed and allowed to stand at room temperature for 5 minutes. Subsequently, 10 μL (0.44 μmol) of an aqueous solution of pdCpA was added and mixed, and allowed to stand for 30 minutes for reaction. After adding 1 mL of diethyl ether and centrifuging at 15000 rpm, the supernatant was removed, and this operation was repeated twice. After removing diethyl ether by a centrifugal evaporator, 50 μL of 0.38% formic acid and 50 μL of acetonitrile were added. The target peak was separated by HPLC, and the solvent was removed by a centrifugal evaporator. A part of the obtained BODIPY FL-pdCpA was hydrolyzed with 0.1 M NaOH, the concentration was calculated from the peak area of the resulting pdCpA, and dissolved in DMSO to 2.2 nmol / μL. The yield was 16 nmol (3.6%).

(7) Synthesis of FAM-AC ₃ -pdCpA
100 μL (0.22 μmol) of AC ₃ -pdCpA was mixed with 10 μL (1 μmol) of a 100 mM DMSO solution of 5-FAM-SE. Subsequently, 110 μL of 0.1M NaHCO ₃ was added and reacted on ice for 1 hour, followed by neutralization by adding 16.5 μL of 1M AcOH. The target peak was separated by HPLC, and the solvent was removed by a centrifugal evaporator. A part of the obtained FAM-AC ₃ -pdCpA was hydrolyzed with 0.1 M NaOH, the concentration was calculated from the peak area of the resulting pdCpA, and dissolved in DMSO to be 2.2 nmol / μL. The yield was 54 nmol (yield 25%).

FIG. 3B shows the structure of FAM-AC ₃ . FAM-AC ₃ -pdCpA is bound to pdCpA via the carboxyl group of the carboxylic acid.

(8) Synthesis of Cy3-AC ₃ -pdCpA
120 μL (0.26 μmol) of AC ₃ -pdCpA was mixed with 12 μL of a 100 mM DMSO solution of Cy3-SE. Subsequently, 132 μL of 0.01M NaHCO ₃ was added and reacted on ice for 5 hours, and then neutralized with 1M AcOH. The target peak was separated by HPLC, and the solvent was removed by a centrifugal evaporator. A part of the obtained Cy3-AC ₃ -pdCpA was hydrolyzed with 0.1M NaOH, and the concentration was calculated from the peak area of the resulting pdCpA, and dissolved in DMSO so as to be 2.2 nmol / μL. The yield was 3.1 nmol (yield 1.2%).

In FIG. 3D, it shows the structure of Cy3-AC _3. Cy3-AC ₃ -pdCpA is bound to pdCpA via the carboxyl group of the carboxylic acid.

(9) Synthesis of Cy5-AC ₃ -pdCpA
80 μL (0.18 μmol) of AC ₃ -pdCpA was mixed with 8 μL of a 100 mM DMSO solution of Cy5-SE. Subsequently, 88 μL of 0.05M NaHCO ₃ was added and reacted on ice for ₃ h, and then neutralized with 1M AcOH. The target peak was separated by HPLC, and the solvent was removed by a centrifugal evaporator. A part of the obtained Cy5-AC ₃ -pdCpA was hydrolyzed with 0.1 M NaOH, and the concentration was calculated from the peak area of the resulting pdCpA, and dissolved in DMSO so as to be 2.2 nmol / μL. The yield was 82 nmol (yield 46%).

In FIG. 3E, it shows the structure of Cy5-AC _3. Cy5-AC ₃ -pdCpA is bound to pdCpA via the carboxyl group of the carboxylic acid.

(10) Synthesis of Alexa488-AC ₃ -pdCpA
20 μL (0.044 μmol) of AC ₃ -pdCpA was mixed with 2 μL of a 100 mM DMSO solution of AlexaFluor488-SE. Subsequently, 22 μL of 0.05M NaHCO ₃ was added and reacted on ice for 3 hours, followed by neutralization with 1M AcOH. The target peak was separated by HPLC, and the solvent was removed by a centrifugal evaporator. A part of the obtained Alexa488-AC ₃ -pdCpA was hydrolyzed with 0.1M NaOH, the concentration was calculated from the peak area of the resulting pdCpA, and dissolved in DMSO to be 2.2 nmol / μL. The yield was 10 nmol (yield 23%).

(11) Synthesis of Biotin-TAMRA-X-AF-pdCpA 1.56 mL (7.8 μmol) of 5 mM DMSO solution of aminophenylalanine-pdCpA (AF-pdCpA) was mixed with 323 μL of 50 mM DMSO solution of TAMRA-X-SE and 167 μL of DMSO. . Subsequently, 2 mL of a 1M Pyridine-HCl aqueous solution (pH 5) was added and reacted at 37 ° C. for 5 hours. The target peak was separated by HPLC, and the solvent was removed by a centrifugal evaporator. A part of the obtained TAMRA-X-AF-pdCpA was hydrolyzed with 0.1M NaOH, the concentration was calculated from the peak area of the resulting pdCpA, and dissolved in DMSO to be 2.2 nmol / μL. The yield was 1.8 μmol (23% yield).

50 μL (0.11 μmol) of TAMRA-X-AF-pdCpA was mixed with 30 μL of 50 mM DMSO solution of Biotin-SE and 30 μL of DMSO. Subsequently, 10 μL of 0.1M NaHCO ₃ was added and reacted on ice for 21 hours, and then neutralized with 1M AcOH. The target peak was separated by HPLC, and the solvent was removed by a centrifugal evaporator. A part of the obtained Biotin-TAMRA-X-AF-pdCpA was hydrolyzed with 0.1M NaOH, the concentration was calculated from the peak area of the resulting pdCpA, and dissolved in DMSO to be 2.2 nmol / μL. It was. The yield was 75 nmol (68% yield).

(12) Synthesis of carboxylic acid-tRNA
5 × Ligation Buffer (275 mM Hepes-Na pH 7.5, 75 mM MgCl ₂ , 16.5 mM DTT, 5 mM ATP) 4 μL, 200 μM tRNA (-CA) (E. coli fMet tRNA with anticodon replaced with CUA And A-deficient, SEQ ID NO: 1) 2.5μL, 2μL of carboxylic acid-pdCpA in DMSO, 0.4μL of 0.1% BSA, 1.2μL of T4 RNA Ligase (25units / μL), 9.9μL of water, mixed at 4 ℃ For 12 hours. After adding 2 μL of 3M AcOK pH 4.5, 66 μL of ethanol was added, mixed gently, and placed at −30 ° C. for 1 hour. After centrifugation at 4 ° C for 15000 rpm for 30 minutes, the supernatant was removed, and 200 µL of 70% ethanol stored at -30 ° C was added, followed by centrifugation at 15000 rpm at 4 ° C for 5 seconds. The supernatant was removed and dried under reduced pressure. Dissolved in 2 μL of 1 mM potassium acetate pH 4.5.

Example 2 Introduction of a carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position to which a labeling substance has been attached (1) Amino group, amide bond and hydroxyl at the α-position to which TAMRA was added Introduction of carboxylic acid having no group into protein N-terminus In reaction solution (10 μL), 55 mM Hepes-KOH (pH 7.5), 210 mM potassium glutamate, 6.9 mM ammonium acetate, 1.7 mM dithiothreitol, 1.2 mM ATP, 0.28 mM GTP, 26 mM phosphoenolpyruvate, 1 mM spermidine, 1.9% polyethylene glycol-8000, 35 μg / mL Folinic acid, 12 mM magnesium acetate, 0.1 mM 20 amino acids, streptavidin mRNA (with the start codon replaced by UAG, sequence Number 2) 1 μL of 8 μg / μL, 2 μL of E. coli extract (Promega) and 1 μL of carboxylic acid-tRNA solution were mixed. The translation reaction was performed at 37 ° C for 1 hour. 1 μL of RNaseA (10 mg / ml) was added to 1 μL of the translation reaction solution and reacted at 37 ° C. for 15 minutes. Then, 9 μL of water and 10 μL of 2 × sample buffer were added and heated at 95 ° C. for 5 minutes. 5 μL of this was applied to 15% SDS-PAGE, and after completion, the sample was observed with a fluorescence scanner (HMBIO-III manufactured by Hitachi Software Engineering, excitation light 532 nm, fluorescence filter 580 nm). When carboxylic acid-tRNA having no amino group, amide bond and hydroxyl group was added to the α-position to which TAMRA was added, a band of streptavidin into which TAMRA was introduced was confirmed. FIG. 7 shows the results of SDS-PAGE. FIG. 8 shows the fluorescence intensity of the band of streptavidin. The sequence of the tRNA used is shown in SEQ ID NO: 1.

(2) Introduction of a carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position to the protein N-terminus with BODIPY FL added
This was carried out in the same manner as in the case of a carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position, to which TAMRA was added. However, the fluorescence scanner was set to excitation light 488 nm and fluorescence filter 530 nm. When carboxylic acid-tRNA having no amino group, amide bond and hydroxyl group was added to the α-position to which BODIPY FL was added, a band of streptavidin into which BODIPY FL was introduced was confirmed. FIG. 9 shows the results of SDS-PAGE. FIG. 10 shows the fluorescence intensity of the band of streptavidin.

(3) Introduction of 3-aminopropionic acid with various fluorescent groups added to the protein N-terminus
This was carried out in the same manner as in the case of a carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position, to which TAMRA was added. However, the settings of the fluorescence scanner were excitation light 488 nm / fluorescence filter 530 nm, excitation light 532 nm / detection filter 605 nm, excitation light 635 nm / detection filter 670 nm. When 3-aminopropionic acid-tRNA added with various fluorescent groups was added, a band of streptavidin into which each fluorescent group was introduced was confirmed. FIG. 11 shows the results of SDS-PAGE.

(4) Introduction of carboxylic acid with TAMRA and biotin added to the protein N-terminus
TAMRA was added using mRNA (SEQ ID NO: 3) in which the start codon of fatty acid binding protein (FABP) to which Flag tag peptide was added was substituted with UAG, and it did not have an amino group, amide bond or hydroxyl group at the α-position. It carried out like the case of carboxylic acid. However, the fluorescence scanner was set to excitation light of 532 nm / detection filter of 605 nm, and SDS-PAGE was also performed when 1 μL of 1 mM streptavidin was added to the electrophoresis sample after purification and after purification. Purification was performed using MagneHis Ni-particles (Promega) and following the recommended protocol. FIG. 12 shows the results of SDS-PAGE. As shown in FIG. 12, amino acids labeled with both biotin and TAMRA were introduced at the N-terminus of the protein. The introduction of biotin was confirmed from the shift of the band position by addition of streptavidin.

(5) Introduction of BODIPY FL-3-aminopropionic acid into the protein N-terminus when a 4-base codon is used as the start codon tRNA (-CA) in which the anticodon of tRNA (-CA) of SEQ ID NO: 1 is replaced with CCCG ( Using SEQ ID NO: 4), BODIPY FL-3-aminopropionic acid-tRNA was prepared according to the synthesis method of carboxylic acid-tRNA. Subsequently, using a mRNA (SEQ ID NO: 5) in which the start codon of the streptavidin mRNA of SEQ ID NO: 2 is substituted with CGGG (SEQ ID NO: 5), a carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position is added. In the same manner as described above, BODIPY FL-3-aminopropionic acid was introduced into the protein N-terminus using a 4-base codon as the start codon. However, the fluorescence scanner was set to excitation light 488 nm and fluorescence filter 530 nm. FIG. 13 shows the results when the start codon is replaced with CGGG and the anticodon of the start tRNA is replaced with CCCG. As shown in FIG. 13, even when the start codon was replaced with CGGG and the anticodon of the start tRNA was replaced with CCCG, a fluorescently labeled amino acid could be introduced at the N-terminus of the protein.

Experimental Example 3 Introduction of fluorescently labeled hydroxy acid into protein N-terminus (1) Synthesis of pN-Boc-aminophenyllactic acid cyanomethyl ester
To 1.5 g (7.1 mmol) of p-nitro-L-phenylalanine, add 10 mL of water, 12 mL of 1 M sulfuric acid aqueous solution, and 22.5 mL of acetone, and add dropwise 10 mL of 2.2 M sodium nitrite aqueous solution over 30 minutes with stirring under ice cooling. The mixture was reacted for 1.5 hours under ice-cooling, and then allowed to react for 16 hours at room temperature. After the reaction, only acetone was removed with an evaporator, ethyl acetate was added, washed twice with water and once with saturated saline. Thereafter, anhydrous sodium sulfate was added for dehydration, and after filtration, the solvent was removed by an evaporator until a small amount of ethyl acetate remained. Isopropyl acetate was added and dried with an evaporator. Hexane was added to suspend the precipitate, and the precipitate was collected by suction filtration. The precipitate was washed with a small amount of isopropyl acetate and a large amount of hexane, and then dried under reduced pressure to obtain 810 mg (3.84 mmol; 54%) of p-nitrophenyllactic acid.

  275 mg of 10% palladium carbon was added to 507 mg (2.40 mmol) of p-nitrophenyl lactic acid, hydrogen substitution was performed, 10 mL of dehydrated ethanol was added, and the mixture was reacted at room temperature for 1.5 hours. The reaction solution was filtered, and the precipitate was washed and collected with methanol and dried with an evaporator to obtain 174 mg (0.96 mmol; 40%) of p-aminophenyllactic acid.

To 36.1 mg (0.20 mmol) of p-aminophenyl lactic acid, add 0.1 M aqueous sodium hydrogen carbonate solution (4.5 mL), 1,4-dioxane (4.5 mL), 3.7 M (Boc) ₂ O (111 μL), and react at room temperature for 3 hours. It was. Remove the solvent with an evaporator, add ethyl acetate, wash once with 5% aqueous potassium hydrogensulfate solution, add anhydrous magnesium sulfate to dehydrate, filter, and dry with an evaporator to obtain 44.6 pN-Boc-aminophenyllactic acid. mg (0.16 mmol; 80%) was obtained.

  To 3.7 mg (13.3 μmol) of p-N-Boc-aminophenyl lactic acid, 230 μL of dehydrated acetonitrile, 111 μL of triethylamine, and 84 μL of chloroacetonitrile were added and reacted at room temperature for 3 hours. Ethyl acetate was added, washed twice with 5% potassium hydrogen sulfate, and the solvent was removed by a centrifugal evaporator. The target peak was collected by HPLC, and the solvent was removed by a centrifugal evaporator to obtain 1.9 mg (5.9 μmol; 44%) of p-N-Boc-aminophenyllactic acid cyanomethyl ester.

(2) Synthesis of p-aminophenyllactic acid-pdCpA (AFL-pdCpA)
To 70.5 μL (6.20 μmol) of pdCpA in DMF, 5.9 mg (18.4 μmol) of pN-Boc-aminophenyllactic acid cyanomethyl ester was added in three portions and reacted at room temperature for 6.5 hours. 1 mL of diethyl ether was added to collect the precipitate, which was further washed twice with diethyl ether and then dried under reduced pressure. The target peak was separated by HPLC, and the solvent was removed by a centrifugal evaporator. Subsequently, 200 μL of trifluoroacetic acid was added and left on ice for 10 minutes to remove the Boc group. After concentration under reduced pressure with a centrifugal evaporator, 1 mL of diethyl ether was added and stirred, and after centrifugation, diethyl ether was removed and dried under reduced pressure with a centrifugal evaporator. A part of the obtained p-aminophenyl lactic acid-pdCpA was hydrolyzed with 0.1M NaOH, the concentration was calculated from the peak area of the resulting pdCpA, dissolved in DMSO to be 2.2 nmol / μL, and p -Aminophenyllactic acid-pdCpA in DMSO solution 697 μL (70%).

(3) Synthesis of pN-BODIPYFL-aminophenyl lactate-pdCpA (BODIPYFL-AFL-pdCpA)
To 180 μL (0.396 μmol) of DMSO solution of p-aminophenyllactic acid-pdCpA, 36 μL (1.8 μmol) of DMSO solution of 50 mM BODIPYFL-SE and 216 μL of 1M pyridine-HCl aqueous solution (pH 5.0) were added and reacted at 37 ° C. I let you. After 1.5 hours, 18 μL (0.9 μmol) of 50 mM BODIPYFL-SE in DMSO was added. Three hours after the start of the reaction, the reaction solution was dispensed into four tubes, 1 mL each of diethyl ether was added and stirred, and the operation of removing the diethyl ether layer after centrifugation was further repeated twice. The target peak was separated by HPLC, and the solvent was removed by a centrifugal evaporator. Part of the obtained pN-BODIPYFL-aminophenyllactic acid-pdCpA was hydrolyzed with 0.1M NaOH, the concentration was calculated from the peak area of the resulting pdCpA, and dissolved in DMSO to be 2.2 nmol / μL. As a result, 68.0 μL (37%) of a DMSO solution of pN-BODIPYFL-aminophenyllactic acid-pdCpA was obtained.

(4) Synthesis of pN-TAMRAX-aminophenyllactic acid-pdCpA (TAMRA-X-AFL-pdCpA)
To 45 μL (99 nmol) of DMSO solution of p-aminophenyllactic acid-pdCpA, 4.5 μL (450 nmol) of DMSO solution of 100 mM TAMRAX-SE, 54 μL of a 1: 1 mixture of DMSO solution of 6M pyridine-HCl and pyridine was added. In addition, the mixture was reacted at 37 ° C. for 12 hours. The operation of adding 1 mL of chloroform to the reaction solution and stirring, and removing the chloroform layer after centrifugation was repeated three more times. The target peak was separated by HPLC, and the solvent was removed by a centrifugal evaporator. A part of the obtained pN-TAMRAX-aminophenyllactic acid-pdCpA is hydrolyzed with 0.1M NaOH, the concentration is calculated from the peak area of the resulting pdCpA, and dissolved in DMSO to be 2.2 nmol / μL. 9.1 μL (20%) of DMSO solution of pN-TAMRAX-aminophenyllactic acid-pdCpA was obtained.

(5) Introduction of fluorescently labeled hydroxy acid into protein Fluorescently labeled hydroxy acid-tRNA was synthesized according to the synthesis method of carboxylic acid-tRNA. Subsequently, in the case of a carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position, wherein TAMRA is added using mRNA obtained by adding SEQ ID NO: 6 to 11 upstream of the N-terminus (Asp1) of streptavidin In the same manner as described above, a fluorescently labeled hydroxy acid was introduced into a protein. However, the settings of the fluorescence scanner were excitation light 488 nm / fluorescence filter 530 nm for BODIPY FL, excitation light 532 nm / detection filter 605 nm, excitation light 635 nm / detection filter 670 nm for TAMRA. FIG. 14 shows the results of introduction of a fluorescently labeled hydroxy acid into T7tag-ACT-streptavidin into a protein. As shown in FIG. 14, it was found that when a fluorescently labeled hydroxy acid was introduced, N-terminal fluorescently labeled streptavidin lacking the T7 tag was obtained.

FIG. 15 shows the results of alkaline hydrolysis by adding NaHCO ₃ to the crude protein obtained in FIG. As shown in FIG. 15, it was found that only N-terminal fluorescently labeled streptavidin lacking the T7 tag was obtained by alkaline hydrolysis.

  FIG. 16 shows the results of introduction of a fluorescently labeled hydroxy acid into a protein when tag peptides of T7tag, RGSHis3, VGV-G, Avi, and Glu-Glu (E.E.) are added upstream of streptavidin. As shown in FIG. 16, in any tag peptide, it was found that when a fluorescently labeled hydroxy acid was introduced, the tag peptide was removed and streptavidin having BODIPY FL-AFL at the N-terminus was obtained.

  The tRNA of the present invention can be used to produce a protein whose N-terminus is specifically modified with a labeling substance such as a fluorescent substance, and the N-terminal modified protein can be used as a labeled protein for various biochemical analyses. .

It is a figure which shows the structure of TAMRA labeling carboxylic acid. It is a figure which shows the structure of BODIPY FL labeling carboxylic acid. It is a figure which shows the structure of the carboxylic acid labeled with various fluorescent substances. It is a figure which shows the structure of the carboxylic acid labeled with TAMRA and biotin. It is a figure which shows the structure of the hydroxy acid labeled with TAMRA and BODIPY FL. pdCpA is a diagram showing a structure of a coupled to form carboxylic acid _{(BODIPY FL-ABC 2 -pdCpA)} . It is a figure which shows the result of SDS-PAGE of streptavidin which introduce | transduced TAMRA labeling carboxylic acid into the N terminal. It is a figure which shows the fluorescence band intensity in SDS-PAGE of streptavidin which introduce | transduced TAMRA labeling carboxylic acid into the N terminal. It is a figure which shows the result of SDS-PAGE of streptavidin which introduce | transduced BODIPY FL labeling carboxylic acid into the N terminal. It is a figure which shows the fluorescence band intensity in SDS-PAGE of the streptavidin which introduce | transduced BODIPY FL labeling carboxylic acid to the N terminal. It is a figure which shows the result of SDS-PAGE of the streptavidin which introduce | transduced various fluorescent label | markers (Alexa488, BODIPY FL, Cy3, Cy5, FAM, and TAMRA) carboxylic acid into the N terminal. It is a figure which shows the result of SDS-PAGE of FABP which introduce | transduced the carboxylic acid labeled with TAMRA and biotin into the N terminal. It is a figure which shows the result of having introduce | transduced BODIPY labeled carboxylic acid into the N terminal using 4 base codons as a start codon. It is a figure which shows the result of SDS-PAGE of the fusion protein which introduce | transduced the hydroxy acid labeled with the fluorescent substance. It is a figure which shows the result of SDS-PAGE of what hydrolyzed the fusion protein which introduce | transduced the hydroxy acid labeled with the fluorescent substance. It is a figure which shows the result of SDS-PAGE when hydroxy acid is introduce | transduced into the fusion protein of various tags and streptavidin.

SEQ ID NOs: 2, 4 to 11

Claims (21)

  Initiation tRNA to which a carboxylic acid having no amino group, amide bond and hydroxyl group is added at the α-position.   The initiating tRNA according to claim 1, wherein a carboxylic acid having no amino group, amide bond and hydroxyl group is added to the α-position and β-position.   A carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position or a carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position and the β-position is modified with at least one labeling substance. The starting tRNA according to 1 or 2.   A carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position or a carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position and the β-position is modified with at least two labeling substances. 3. The starting tRNA according to 3.   The initiation tRNA according to claim 3 or 4, wherein the modification is a label with a fluorescent substance, a quencher substance, a cross-linking substance, digoxigenin, dinitrophenyl, cholesterol, desthiobiotin or biotin.   The starting tRNA according to claim 1, wherein acetic acid, pyroglutamic acid, palmitoyl acid or myristoyl acid is added as a carboxylic acid.   The start tRNA according to any one of claims 1 to 6, wherein AUG is recognized as a start codon.   The initiation tRNA according to any one of claims 1 to 6, which recognizes UAG, UAA or UGA as an initiation codon.   The start tRNA according to any one of claims 1 to 6, which recognizes a three-base codon other than AUG, UAG, UAA and UGA as a start codon.   The start tRNA according to any one of claims 1 to 6, wherein a 4-base codon is recognized as a start codon.   A carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position, or an amino group, amide bond and hydroxyl group at the α-position and β-position using the initiation tRNA according to any one of claims 1 to 10 A method for producing a protein having a carboxylic acid introduced at the N-terminus by specifically introducing a carboxylic acid having no carboxylic acid into the N-terminus of the protein.   A carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position or an amino group at the α-position and the β-position, using the starting tRNA modified with the labeling substance according to any one of claims 3 to 10. A method for producing a protein having a N-terminal modified with a labeling substance by specifically introducing a carboxylic acid having no group, amide bond and hydroxyl group and labeled with a labeling substance into the N-terminal of the protein .   A DNA or mRNA encoding a protein, which has a 4-base codon as a codon corresponding to the N-terminal amino acid of the protein, and a 3-base codon other than AUG, UAG, UAA, UGA or AUG, UAG, UAA and UGA as a start codon Using the DNA or mRNA containing as a template, further using the start tRNA according to any one of claims 1 to 10 having a sequence corresponding to the start codon of the DNA or mRNA at the anticodon site, Performs protein biosynthesis, specifically carboxylic acid without amino group, amide bond and hydroxyl group at α-position or carboxylic acid without amino group, amide bond and hydroxyl group at α-position and β-position to the N-terminus of protein A method for producing a protein having a carboxylic acid introduced at the N-terminus.   A DNA or mRNA encoding a protein, which has a 4-base codon as a codon corresponding to the N-terminal amino acid of the protein, and a 3-base codon other than AUG, UAG, UAA, UGA or AUG, UAG, UAA and UGA as a start codon A start tRNA that is modified with the labeling substance according to any one of claims 3 to 10 using a DNA or mRNA as a template, and a sequence corresponding to the start codon of the DNA or mRNA at an anticodon site. Carry out protein biosynthesis using the starting tRNA with carboxylic acid that does not have an amino group, amide bond and hydroxyl group labeled with a labeling substance, or an amino group, amide bond and hydroxyl group at the α and β positions. A method for producing a protein in which the N-terminus is modified with a labeling substance by specifically introducing a carboxylic acid having no saccharide to the N-terminus of the protein.   A carboxylic acid having no amino group, amide bond and hydroxyl group at the α-position, or an amino group, amide bond and hydroxyl group at the α-position and β-position, produced by the method according to claim 11. A protein having a carboxylic acid having no carboxylic acid at the N-terminus, wherein the carboxylic acid is labeled with a labeling substance or not labeled.   A tRNA added with an α-hydroxy acid having a hydroxyl group at the α-position and modified with at least one labeling substance.   The tRNA according to claim 16, which is modified with at least two labeling substances.   The tRNA to which an α-hydroxy acid is added according to claim 16 or 17, wherein the modification is labeling with a fluorescent substance, a quencher substance, a cross-linking substance, digoxigenin, dinitrophenyl, cholesterol, desthiobiotin, or biotin.   The tRNA to which an α-hydroxy acid according to any one of claims 16 to 18 recognizes a 4-base codon, a stop codon, an artificial codon, or a codon assigned with an amino acid in nature.   A fusion DNA or mRNA encoding the first protein and the second protein or peptide is used as a template, and an α-hydroxy acid modified with the labeling substance according to any one of claims 16 to 19 is further added. A fusion protein in which the N-terminus of the first protein and the C-terminus of the second protein or peptide are bound using the tRNA, and is the N-terminal site of the first protein and the second protein or peptide A fusion protein in which a hydroxy acid modified with a labeling substance is introduced next to the C-terminus of the first protein, between the N-terminal hydroxy acid of the first protein and the C-terminal amino acid of the second protein or peptide Hydrolyzing the ester bond and isolating a first protein introduced with a hydroxy acid modified with a labeling substance at the N-terminus, A method for producing a protein labeled with a labeling substance.   21. The method for producing a protein having an N-terminal labeled with a labeling substance according to claim 20, wherein the second protein or peptide is a tag peptide.

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