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3-pyrazolyl tyrosine translation system and application thereof

A translation system, tyrosine technology, applied in green fluorescent protein mutants, 3-pyrazolyl tyrosine translation system, translation system, application of green fluorescent protein mutants, aminoacyl-tRNA synthetase mutants, In the field of 3-pyrazolyl tyrosine-2-amino-3-phenyl) propionic acid, it can solve the problems of inability to deeply understand the electron transfer mechanism and the lack of adding electron acceptors.

Active Publication Date: 2014-02-12
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of effective methods for adding electron acceptors to specific sites in target proteins, it is difficult to gain insight into the electron transfer mechanism of GFP in vivo.

Method used

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  • 3-pyrazolyl tyrosine translation system and application thereof
  • 3-pyrazolyl tyrosine translation system and application thereof
  • 3-pyrazolyl tyrosine translation system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1: the chemical synthesis of pyTyr (see figure 1 with figure 2 )

[0055] Add 3-iodotyrosine (2.46g, 8mmol, purchased from Shanghai Gil Biochemical Company) in a 50ml round bottom flask, dissolve in 20ml 10% NaOH aqueous solution, and take t-Boc anhydride (1.92g, 8.8mmol, (purchased from Beijing Zhongsheng Huateng Co., Ltd.) was dissolved in 20ml THF, and added dropwise to 3-iodotyrosine NaOH solution, and stirred overnight at room temperature. After stopping the reaction, add an appropriate amount of hydrochloric acid to adjust the pH value to 6.5-7.0, then extract with ethyl acetate, collect the ethyl acetate phase and carry out rotary evaporation to obtain 2.94g of Boc-L-3-iodotyrosine acid.

[0056] Take a clean 50ml three-necked bottle, add pyrazole (0.34g, 5mmol, purchased from Sigma Company), anhydrous Cs 2 CO 3 (1.92g, 10mmol, purchased from Tianjin Alfa Aesar Company), CuI (0.019g, 0.1mmol, as a catalyst), Boc-L-3-iodotyrosine (2.03g, 5mmol) an...

Embodiment 2

[0058] Example 2: Evolution of pyTyr-specific aminoacyl-tRNA synthetases

[0059] For site-specific insertion of pyTyr in the gene, it is necessary to introduce an aminoacyl-tRNA synthetase / tRNA orthogonal pair derived from the amber suppressor of Methanococcus jannaschii into the E.coli host cell used. Tyrosyl 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 laboratory of Peter G. Schultz, 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, ...

Embodiment 3

[0062] Example 3: Expression of pyTyr-myoglobin, pyTyr-green fluorescent protein and identification by mass spectrometry

[0063] The orthogonal tRNA (SEQ ID NO: 1) and the screened pyTyrRS (SEQ ID NO: 3) were respectively constructed on the pEVOL vector (purchased from Peter G. pbad-myoglobin (4TAG) or pET-green fluorescent protein (151TAG) expression plasmid (the plasmid was purchased from Peter G.Schultz laboratory of Scripps Research Institute, USA) (wherein the nucleotide sequence of myoglobin is SEQ ID NO: 5. The nucleotide sequence of green fluorescent protein is SEQ ID NO: 10) in DH10B cells (purchased from Quanshijin Company). Pick a single clone and grow to OD at 37°C 600 When it was approximately equal to 1.1, 0.5mM pyTyr, 1mM IPTG and 0.2% arabinose (purchased from Sigma) were added to the LB medium to culture the cells, and the control did not add pyTyr. After 6-8 hours, the bacteria were harvested, and the protein was purified by Ni-NTA, and analyzed by SDS-PAG...

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Abstract

The invention relates to a 3-pyrazolyl tyrosine translation system and an application thereof. The invention relates to an aminoacyl-tRNA synthetase mutant, and an amino acid sequence contained in the aminoacyl-tRNA synthetase mutant is selected from a group consisting of amino acids as shown in SEQ (sequence) ID (identity) NO: 3 and conservative variants thereof. The invention provides a 3-pyrazolyl tyrosine translation system inserting 3-pyrazolyl tyrosine (pyTyr) into target proteins in a fixed-point specific manner by utilizing the pairing of orthogonal tRNA (transfer ribonucleic acid) and orthogonal aminoacyl-tRNA synthetase and a method for inserting the 3-pyrazolyl tyrosine into the target proteins in a fixed-point specific manner by utilizing the translation system. The 3-pyrazolyl tyrosine translation system contains (i) the 3-pyrazolyl tyrosine; (ii) the orthogonal aminoacyl-tRNA synthetase; (iii) the orthogonal tRNA, wherein the orthogonal aminoacyl-tRNA synthetase uses the 3-pyrazolyl tyrosine to perform priority aminoacylation on the orthogonal tRNA; and (iv) nucleic acid encoding the target proteins, wherein the nucleic acid contains at least one selector codon which is specifically identified 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 acids shown in SEQ ID NO: 3 and their conservative variants. The present invention also relates to a kind of 3-pyrazolyltyrosine ((S)-2-amino-3-(4-hydroxyl-3-(1H-pyrazol-1-yl)phenyl)propionic acid, abbreviated as pyTyr) and a translation system comprising it. More specifically, the present invention relates to a 3-pyrazolyltyrosine translation system for site-specific insertion of 3-pyrazolyltyrosine into target proteins using orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases and their pairings, And a method for site-specific insertion of 3-pyrazolyltyrosine in a target protein by using the translation system. The present invention also relates to mutant proteins containing 3-pyrazolyltyrosine produced by this translation system ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N1/21C12N15/11C12P21/00C12P21/02C07K14/00C07D231/12
CPCC07D231/12C07K14/43595C12N9/93C12P21/02C12Y601/01
Inventor 王江云刘晓红李家松董建树胡诚龚为民江欢欢
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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