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3-Methylthiotyrosine Translation System and Its Application

A technology of methionine, translation system, applied in the direction of carbon-carbon lyase, enzyme, biochemical equipment and method, etc., can solve problems such as inability to detect

Active Publication Date: 2016-03-30
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nevertheless, the exact role of this cofactor in enzymatic catalysis remains largely unknown, and the bottleneck limiting its research is the inability to probe the structure of this post-translational modification by traditional site-directed mutagenesis

Method used

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  • 3-Methylthiotyrosine Translation System and Its Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Embodiment 1: the biocatalytic synthesis of MtTyr ( Figure 1-Figure 4 )

[0078] The present invention adopts the method of biological enzyme catalysis, utilizes tyrosine phenol lyase (TPL) cloned from Citrobacter freundii (ATCC8090, purchased from the American Type Culture Collection (ATCC)) to catalyze 2-hydroxybenzenesulfide Synthesis of 3-methylthiotyrosine from ether, the catalytic reaction formula is shown in figure 1 .

[0079] However, the experimental results showed that wild-type TPL could not catalyze the production of 3-methylthiotyrosine. Therefore, the present inventors analyzed the crystal structure figure of TPL ( figure 2 A), select 448-position phenylalanine, 36-position phenylalanine and 288-position methionine, introduce NNK mutation (N=A+T+C+G; K=T+G), and construct pEt - TPL mutant library for directed evolution of TPL. 96 single clones were selected from the mutant library, cultured overnight in a 96-well plate, lysozyme was added to lyse t...

Embodiment 2

[0083] Example 2: Evolution of MtTyr-specific aminoacyl-tRNA synthetases

[0084] In order to site-specifically insert MtTyr into the gene, it is necessary to introduce an aminoacyl-tRNA synthetase / tRNA orthogonal pair in the E. coli host cell used. Aminoacyl tRNA (MjtRNA CUA Tyr ) / tyrosyl tRNA synthetase (MjTyrRS, wild type, its amino acid sequence is SEQ ID NO: 2) pair. The MjTyrRS mutation library was constructed in the kanamycin-resistant pBK plasmid (purchased from the PeterG. Schultz laboratory of Scripps Research Institute, USA), and located between the promoter and terminator of E. coli glutamine synthetase on the plasmid. The synthetic enzyme mutation library used is the pBk-lib-jw1 library, and the construction method of the mutation library is: select 6 sites (Tyr32, Leu65, Phe108, Gln109, Asp158, and Leu162) on the MjTyrRS gene and introduce NNK mutation ( N=A+T+C+G; K=T+G), the other 6 sites (Ile63, Ala67, His70, Tyr114, Ile159, Val164) were either randomly mut...

Embodiment 3

[0088] Example 3: Expression of MtTyr-myoglobin and identification by mass spectrometry

[0089] Orthogonal tRNA (SEQ ID NO: 1) and the nucleotide sequence (4TAG or 33TAG) (SEQ ID NO: 5 and 7) encoding myoglobin were constructed on the pBAD vector (purchased from PeterG. The screened nucleotide sequence encoding MtTyrRS (SEQ ID NO: 3) was constructed on the pBK vector (purchased from the laboratory of Peter G. Schultz, Scripps Research Institute, USA), and then co-transformed into DH10B cells (purchased from Quanshijin Company). Pick a single clone and grow to OD at 37°C 600 When it was approximately equal to 0.5, 1 mM MtTyr and 0.2% arabinose (purchased from sigma company) were added to the LB medium to culture the cells, and the control did not add MtTyr. After 6-8 hours, the bacteria were harvested, and the protein was purified by Ni-NTA, and analyzed by SDS-PAGE electrophoresis ( Figure 6 A).

[0090] We found that the full-length myoglobin can only be purified in the ...

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Abstract

The invention relates to an aminoacyl-tRNA synthetase mutant. The amino acid sequence of the aminoacyl-tRNA synthetase mutant is selected from a group consisting of an amino acid sequence shown as SEQ ID NO: 4 and conservative variants of the amino acid sequence shown as SEQ ID NO: 4, wherein the conservative variants have the same enzymatic activity as the amino acid sequence shown as SEQ ID NO: 4. The invention provides a 3-methylthio tyrosine translation system. The 3-methylthio tyrosine translation system comprises (i) 3-methylthio tyrosine; (ii) the orthogonal aminoacyl-tRNA synthetase; (iii) an orthogonal tRNA, wherein the orthogonal aminoacyl-tRNA synthetase has a priority of aminoacylating the orthogonal tRNA by using the 3-methylthio tyrosine; (iv) a nucleic acid encoding a target protein, wherein the nucleic acid comprises at least one selecting codon specifically recognized by the orthogonal tRNA.

Description

technical field [0001] The invention belongs to the field of biochemistry. Specifically, the present invention provides an aminoacyl-tRNA synthetase mutant, which contains an amino acid sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 4 and the conservative variant of the amino acid sequence shown in SEQ ID NO: 4, said The conservative variant has the same enzymatic activity as the amino acid sequence shown in SEQ ID NO:4. The present invention also relates to a 2-amino-3-(4-hydroxyl-3-(methylthio)phenyl)propionic acid (abbreviated as 3-methylthiotyrosine, abbreviated as MtTyr) translation system. More specifically, the present invention relates to a 3-methylthiotyrosine translation system that utilizes the pairing of an orthogonal tRNA and an orthogonal aminoacyl-tRNA synthetase to specifically insert 3-methylthiotyrosine into a target protein, and utilizes the A method for site-specific insertion of 3-methylthiotyrosine in a target...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N1/21C12N9/02C12N9/88C12P21/00
CPCC07K14/805C12N9/88C12N9/93C12P21/02C12Y401/99002C12Y601/01
Inventor 王江云周庆胡美荣张维姜丽柯莎周娟作江欢欢
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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