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3-chlorinated tyrosine translation system and application thereof

A technology of chlorotyrosine and translation system, applied in the direction of application, use of vectors to introduce foreign genetic material, biochemical equipment and methods, etc., can solve problems such as not suitable for labeling lysosomes

Active Publication Date: 2013-01-23
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although red fluorescent protein dsRed pKa 4.3 (Baird GS, Zacharias DA, Tsien RY.Proc Natl Acad Sci US A 2000,97 (22): 11984-11989.), cyan fluorescent protein ECFP pKa 4.7 (Patterson G, Day RN, Piston D.J Cell Sci 2001, 114 (Pt5): 837-838.), but the currently used green fluorescent protein EGFP pKa 6.0 and yellow fluorescent protein YFP pKa 5.6 are not suitable for labeling acidic organelles such as lysosomes, but for studying intracellular Protein-protein interactions, as well as the dynamics of multiple proteins, often require a variety of fluorescent proteins of different colors to label

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Evolution of 3-Cl-Tyr-specific aminoacyl-tRNA synthetases

[0054] In order to site-specifically insert 3-Cl-Tyr into the gene, it is necessary to introduce an aminoacyl-tRNA synthetase / tRNA orthogonal pair into the E.coli host cell used, which is derived from Methanococcus jannaschii jannaschii) Amber suppresses tyrosyl tRNA (MjtRNA CUA Tyr ) / tyrosyl tRNA synthetase (MjTyrRS, wild type, its amino acid sequence is SEQ ID NO: 12) 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 ...

Embodiment 2

[0058] Example 2: Expression of 3-Cl-Tyr-myoglobin and identification by mass spectrometry

[0059] The orthogonal tRNA (SEQ ID NO: 1) and the screened aminoacyl-tRNA synthetase mutant 1 (SEQ ID NO: 5) were respectively constructed into the pEVOL vector (purchased from the laboratory of Peter G. Schultz, Scripps Research Institute, USA) Then co-transformed into pbad-myoglobin (4TAG) containing pbad-myoglobin (4TAG) (this plasmid was purchased from the laboratory of Peter G.Schultz of the American Scripps Research Institute) (wherein the nucleotide sequence of myoglobin (4TAG) is SEQ ID NO: 8) 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 3-Cl-Tyr (purchased from Shanghai Jier Biochemical Company) and 0.2% arabinose (purchased from sigma Company) were added to the LB medium to culture the cells, and the control did not add 3-Cl-Tyr. After 6-8 hours, the bacteria were harvested, and ...

Embodiment 3

[0061] Example 3: Expression of ApoA1 inserted with 3-Cl-Tyr and its identification by mass spectrometry

[0062] AopA1 (nucleotide sequence is SEQ ID NO: 9) was constructed on the pet24a vector (purchased from Novagen), and orthogonal tRNA (SEQ ID NO: 1) and the screened aminoacyl-tRNA synthetase mutant 1 (SEQ ID NO: 5) were respectively constructed on the pEVOL vector (purchased from the laboratory of Peter G. Schultz, Scripps Research Institute, USA). TAG was introduced at position 192 of ApoA1 by overlapping PCR method. Co-transform pEVOL-tRNA, pEVOL-3-Cl-TyrRS and pet24a-ApoA1-192TAG into BL21(DE3) cells (purchased from Quanshijin Company), and pick a single clone into 2YT medium (each liter medium contains 16g pancreatic Peptone, 5g yeast powder, 5gNaCl), plus corresponding antibiotics (kanamycin 50ug / ml tetracycline 10ug / ml). When cells grow to OD 600When it is about 1.0, add 1 mM 3-Cl-Tyr, 0.5 mM IPTG, 0.2% arabinose, and continue culturing at 37°C for 6-8 hours. H...

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Abstract

The invention relates to an aminoacyl-tRNA (transfer ribonucleic acid) synthetase mutant, wherein the amino acid sequence contained therein is selected from a group composed of the amino acids shown by the SEQ ID No. 2, 3 and 4 and the conservative mutants thereof. The invention provides a 3-chlorinated tyrosine translation system doping 3-chlorinated tyrosine into the target protein by use of the orthogonal tRNA, orthogonal aminoacyl-tRNA synthetase and the pairing thereof, and a method for doping the 3-chlorinated tyrosine into the target protein by use of the translation system. The 3-chlorinated tyrosine translation system comprises (i) 3-chlorinated tyrosine, (ii) orthogonal aminoacyl-tRNA synthetase, (iii) orthogonal tRNA and (iv) nucleic acid coding the target protein, wherein the orthogonal aminoacyl-tRNA synthetase performs aminoacylation of the orthogonal tRNA by use of the 3-chlorinated tyrosine by priority; and the nucleic acid contains at least one selection coder for specificity identification of 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 amino acid sequences selected from the group consisting of amino acids shown in SEQ ID NO: 2, 3, 4 and their conservative variants. The present invention also relates to a 3-chlorotyrosine (3-Cl-Tyr) translation system. More specifically, the present invention relates to a 3-chlorotyrosine translation system that utilizes orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and their pairings to incorporate 3-chlorotyrosine into target proteins, and utilizes the A method for the incorporation of 3-chlorotyrosine into a protein of interest by the translation system described above. The present invention also relates to 3-chlorotyrosine-containing muteins produced using this translation system and this method. Background technique [0002] Active substances produced in the state of oxidative stress in or...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N1/21C12N15/63
Inventor 王江云张维江欢欢
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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