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3-imidazolyl tyrosine translation system and use thereof

A translation system, imidazole-based technology, applied in the field of biochemistry, can solve the problem that no one knows the exact function

Active Publication Date: 2013-07-24
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, what exactly is the role of CuB in binding and activating oxygen? How is the decrease in oxygen concentration accompanied by the delivery or uptake of protons in the heme-copper binding center? High-resolution X-ray structures reveal that post-translational modifications of HCO form a covalent cross-link between tyrosine C6 and the adjacent histidine Nε2, and that in bacteria and mammals, the Tyr-His covalent The cross-linked structures are all conserved, confirming that they must have important functions, but no one knows the exact function of the structure

Method used

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  • 3-imidazolyl tyrosine translation system and use thereof
  • 3-imidazolyl tyrosine translation system and use thereof
  • 3-imidazolyl tyrosine translation system and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Embodiment 1: the chemical synthesis of imiTyr ( figure 1 , figure 2 )

[0068] In the 50ml three-necked flask, add imidazole (0.34g, 5mmol, purchased from sigma company), anhydrous Cs2CO3 (1.92g, 10mmol, purchased from Tianjin Alfa Aesar company), CuI (0.019g, 0.1mmol), Boc-L- 3-iodotyrosine (Chinese name Boc-L-3-iodotyrosine) (2.03g, 5mmol, purchased from Shanghai Jier Biochemical Company) and 8ml of anhydrous DMF (purchased from Beijing Bailingwei Company). Under the condition of nitrogen protection, stir and reflux for 18 hours. After cooling, it was extracted with ethyl acetate and distilled water, and the aqueous phase was separated and purified by preparative HPLC (separation column YMC AA12S052503WT, purchased from Huideyi Company, flow rate 12ml / min). Yield 50%. MS: m / z: 248[M+H]+; 1H-NMR (600MHz, DMSO-d6): δ11.20(s, 1H), 9.32(s, 1H), 8.46(s, 2H), 7.99( s, 1H), 7.87 (s, 1H), 7.52 (s, 1H), 7.33 (d, 1H), 7.2 (d, 1H), 3.53 (dd, 1H), 3.19 (m, 2H).

[0069] U...

Embodiment 2

[0070] Example 2: Evolution of imiTyr-specific aminoacyl-tRNA synthetases

[0071] For site-specific insertion of imiTyr in the gene, it is necessary to introduce an aminoacyl-tRNA synthetase / tRNA orthogonal pair in the E.coli host cell used, which is derived from the amber suppressor of Methanococcus jannaschii Tyrosyl tRNA (MjtRNACUA Tyr ) / tyrosyl tRNA synthetase (MjTyrRS, wild type, its amino acid sequence is SEQ ID NO: 5) 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, ...

Embodiment 3

[0074] Example 3: Expression of imiTyr-myoglobin and identification by mass spectrometry

[0075] The orthogonal tRNA (SEQ ID NO: 1) and the screened imiTyrRS (SEQ ID NO: 2) were respectively constructed on the pEVOL vector (purchased from Peter G. pbad-myoglobin (4TAG) (the plasmid was purchased from the laboratory of Peter G. Schultz of the Scripps Research Institute in the United States) (the nucleotide sequence of myoglobin is SEQ ID NO: 4) of DH10B cells (purchased from Whole Gold company). Pick a single clone and grow to OD at 37°C 600 When it was approximately equal to 0.5, 1 mM imiTyr (purchased from Shanghai Jier Biochemical Co., Ltd.) and 0.2% arabinose (purchased from sigma Co.) were added to the LB medium to culture the cells, and the control did not add imiTyr. After 6-8 hours, the bacteria were harvested, and the protein was purified by Ni-NTA, and analyzed by SDS-PAGE electrophoresis ( Figure 4 A).

[0076] We found that the full-length myoglobin can be pur...

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Abstract

The invention relates to an aminoacyl-tRNA synthetase mutant. The aminoacyl-tRNA synthetase mutant has an amino acid sequence composed of amino acids shown in the formula of SEQ ID NO.2 and their conservative mutants. The invention provides a 3-imidazolyl tyrosine translation system for specific and fixed-point insertion of 3-imidazolyl tyrosine (imiTyr) into a target protein by utilization of an orthogonal tRNA, an orthogonal aminoacyl-tRNA synthetase and their complex, and also provides a method for specific and fixed-point insertion of the 3-imidazolyl tyrosine into the target protein by the 3-imidazolyl tyrosine translation system. The 3-imidazolyl tyrosine translation system comprises 1), 3-imidazolyl tyrosine, 2), the orthogonal aminoacyl-tRNA synthetase, 3), the orthogonal tRNA, wherein the orthogonal aminoacyl-tRNA synthetase is obtained by preferential aminoacylation of the orthogonal tRNA by the 3-imidazolyl tyrosine, and 4), a nucleic acid for encoding a target protein, wherein the nucleic acid contains at least one selection codons specifically recognized by the orthogonal tRNA. The invention also relates to a method for specific and fixed-point insertion of 3-imidazolyl tyrosine in Myoglobin (Mb) so that the Myoglobin has oxidase activity, and a use of the method.

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 amino acids shown in SEQ ID NO: 2 and their conservative variants. The present invention also relates to a kind of 3-imidazolyltyrosine ((S)-2-amino-3-(4-hydroxyl-3-(1H-imidazol-1-yl)phenyl)propionic acid, abbreviated as imiTyr) translation system. More specifically, the present invention relates to a 3-imidazolyltyrosine translation system that utilizes orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and their pairings to specifically insert 3-imidazolyltyrosine into target proteins, and utilizes The method for site-specific insertion of 3-imidazolyltyrosine in the target protein by the translation system. The invention also relates to mutant proteins containing 3-imidazolyltyrosine produced by this translation system and this method, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12P21/02C12N1/21C07K14/805C12R1/19
Inventor 王江云刘晓红胡诚江欢欢
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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