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3-methylthio tyrosine translation system and application thereof

A technology of methionine and amino acid, which is applied in the field of biochemistry and can solve problems such as inability to detect

Active Publication Date: 2014-05-28
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-methylthio tyrosine translation system and application thereof
  • 3-methylthio tyrosine translation system and application thereof
  • 3-methylthio tyrosine translation system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1: Biocatalytic synthesis of MtTyr ( Figure 1-Figure 4 )

[0078] The present invention adopts the method of biological enzyme catalysis, using tyrosine phenol lyase (TPL) cloned from Citrobacter Freundii (ATCC 8090, purchased from the American Type Culture Collection (ATCC)) to catalyze 2-hydroxybenzene Synthesis of 3-Methylthiotyrosine from thioether, see the catalytic reaction formula figure 1 .

[0079] However, experimental results show that wild-type TPL cannot catalyze the production of 3-methylthiotyrosine. Therefore, the inventors analyzed the crystal structure diagram 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), construct pEt -TPL mutation library for directed evolution of TPL. 96 single clones were selected from the mutant library and cultured overnight in a 96-well plate. Lysozyme was added to lyse the cells, and then 2-hydroxyphenyl sulfide, ...

Embodiment 2

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

[0084] In order to specifically insert MtTyr into the gene, it is necessary to introduce the aminoacyl-tRNA synthetase / tRNA orthogonal pair into the E.coli host cell used. This orthogonal pair is derived from Methanococcus jannaschii amber inhibition Tyrosyl tRNA (MjtRNA CUA Tyr ) / Tyrosyl tRNA synthetase (MjTyrRS, wild type, whose amino acid sequence is SEQ ID NO: 2) pair. The MjTyrRS mutation library was constructed in a kanamycin-resistant pBK plasmid (purchased from the Peter G. Schultz laboratory of the Scripps Research Institute, USA), located between the promoter and terminator of E. coli glutamine synthetase on the plasmid. The synthetase mutation library used is the pBk-lib-jw1 library, and the method for constructing the mutation library is to select 6 sites (Tyr32, Leu65, Phe108, Gln109, Asp158, and Leu162) on the MjTyrRS gene to introduce NNK mutations N=A+T+C+G; K=T+G), the other 6 sites (Ile...

Embodiment 3

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

[0089] The orthogonal tRNA (SEQ ID NO: 1) and the nucleotide sequence (4TAG or 33TAG) encoding myoglobin (SEQ ID NO: 5 and 7) were constructed into the pBAD vector (purchased from the American Scripps Research Institute Peter G. Schultz) Laboratory), the selected nucleotide sequence encoding MtTyrRS (SEQ ID NO: 3) was constructed on pBK vector (purchased from the Peter G. Schultz laboratory of the Scripps Research Institute, USA), and then co-transformed into DH10B cells (purchased From the full gold company). Pick a single clone and culture it to OD at 37°C 600 When approximately equal to 0.5, 1mMMtTyr 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 collected, the protein was purified by Ni-NTA, and analyzed by SDS-PAGE electrophoresis ( Image 6 A).

[0090] We found th...

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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 aminoacyl-tRNA synthetase mutants, which contain an amino acid sequence selected from the amino acid sequence shown in SEQ ID NO: 4 and conservative variants of the amino acid sequence shown in SEQ ID NO: 4 Group, the conservative variant has the same enzyme activity as the amino acid sequence shown in SEQ ID NO:4. The invention also relates to a translation system of 2-amino-3-(4-hydroxy-3-(methylthio)phenyl)propionic acid (3-methylthiotyrosine for short, MtTyr for short). More specifically, the present invention relates to a 3-methylthiotyrosine translation system that uses orthogonal tRNA and orthogonal aminoacyl-tRNA synthetase pairing to site-specifically insert 3-methylthiotyrosine into a target protein, and uses all The translation system is a method for site-specific insertion of 3-methylthiotyrosine into the target protein. The present invention als...

Claims

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Application Information

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Patent Type & Authority Applications(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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