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3,5-Difluorotyrosine translation system and its application

A technology of difluorotyrosine and translation system, which is applied to the analysis by nuclear magnetic resonance, the introduction of foreign genetic material and enzymes by using vectors, and can solve the problem of low tyrosine phosphorylation ratio

Active Publication Date: 2016-08-17
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, tyrosine phosphorylation accounts for a very low proportion of all phosphorylation modifications in the cell
About 10% of cellular proteins are covalently modified by phosphorylation, but only one tyrosine group is modified in every 100 protein phosphorylation modifications

Method used

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  • 3,5-Difluorotyrosine translation system and its application
  • 3,5-Difluorotyrosine translation system and its application
  • 3,5-Difluorotyrosine translation system and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Embodiment 1: The chemical synthesis of o-phospho-3,5-difluorotyrosine (pF2Y)

[0088] Take 100mM phosphorus oxychloride, 100mM 3,5-difluorotyrosine (purchased from Shanghai Jier Biochemical Company) and 500mM sodium hydroxide, dissolve to 5mL, and then stir at room temperature for 1h. The aqueous phase was collected and separated and purified by HPLC to obtain a white powder with a yield of 10%. (YMC AA12S052503WT column, 12ml / min flow rate, from10%to90%CH3CN, 0.1%TFA(w / v) in water, over the course of 30 min).MS: m / z: 298[M+H]+; 1H-NMR (600 MHz, D2O): 7.03 (d, 2H) 4.01 (dd, 1H) 3.21 (m, 2H).

[0089] Unless otherwise specified, the chemical reagents required for the above synthesis reactions were purchased from Beijing Chemical Plant and were of analytical grade or above.

Embodiment 2

[0090] Example 2: Evolution of F2Y-specific aminoacyl-tRNA synthetases

[0091] In order to site-specifically insert F2Y 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 Y ) / tyrosyl tRNA synthetase (MjYRS, wild type, its amino acid sequence is SEQ ID NO: 2) pair. The MjYRS 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 mutant library used is the pBk-lib-jw1 library, and the construction method of the mutant library is: select 6 sites (Y32, Leu65, Phe108, Gln109, Asp158, and Leu162) on the MjYRS gene to introduce NNK mutation ( N=A+T+C+G; K=T+G), the other 6 sites (Ile63, Ala67, His70, Y114, Ile159, Vall64) were either randomly mutated to Gly or remaine...

Embodiment 3

[0096] Example 3: Analysis of aminoacylation in vitro

[0097] In order to verify the high efficiency and fidelity of F2YRS integration of F2Y in the target protein, we performed an in vitro aminoacylation assay. Take 50mM sodium chloride, 20mM magnesium chloride, 4mM dithiothreitol, 2mM ATP, 10μM Tyr tRNA, 3μM F2YRS and 2mM tyrosine or F2Y, dissolve in 20mM Tris buffer, pH 8.0, and incubate at 37°C for 1h. The reaction solution was analyzed by 24h acid urea polyacrylamide gel electrophoresis, and the results were as follows: figure 2 As shown in A, F2YRS can only integrate F2Y, but not tyrosine.

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Abstract

The invention relates to an aminoacyl-tRNA synthetase mutant which is orthogonal aminoacyl-tRNA synthetase. The amino acid sequence of the synthetase is selected from a group composed of an amino acid sequence presented in SEQ ID NO:4 and conservative variants of the amino acid sequence presented in SEQ ID NO:4, and the conservative variants have enzyme activity the same as the amino acid sequence presented in SEQ ID NO:4. The invention further provides a 3,5-difluoro-tyrosine translation system which comprises (i) 3,5-difluoro-tyrosine, (ii) the orthogonal aminoacyl-tRAN synthetase, (iii) orthogonal tRNA and (iv) nucleic acid of encoded target protein, wherein the orthogonal aminoacyl-tRAN synthetase carries out prior aminoacylation on the orthogonal tRNA through the 3,5-difluoro-tyrosine, and the nucleic acid contains at least one selective codon for specific recognition of 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 is an orthogonal aminoacyl-tRNA synthetase, which contains an amino acid sequence selected from the amino acid sequence shown in SEQ ID NO: 4 and SEQ ID A group consisting of conservative variants of the amino acid sequence shown in NO:4, the conservative variants having the same enzymatic activity as the amino acid sequence shown in SEQID NO:4. The present invention also relates to a 3,5-difluorotyrosine (abbreviated as F2Y) translation system. More specifically, the present invention relates to site-specific insertion of 3,5-difluorotyrosine into 3,5-difluorotyrosine of a target protein using a pair of orthogonal tRNA, orthogonal aminoacyl-tRNA synthetase A translation system, and a method for site-specifically inserting 3,5-difluorotyrosine into a target protein using the translation system. The invention also ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N1/21C12N9/12C12P21/00G01N24/08
CPCC12N9/1205C12N9/93C12N15/70C12Y601/01G01N24/087
Inventor 王江云李发慧李家松龚为民江欢欢
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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