8-hydroxyquinoline alanine translation system and application thereof

A technology of hydroxyquinoline alanine and translation system, applied in the field of biochemistry, can solve problems such as increasing workload

Active Publication Date: 2014-09-24
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the same time, since false positive clones often appear during the screening process, we need to verify their ability to insert unnatural amino acids through further experiments in turn, which virtually increases a lot of workload

Method used

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  • 8-hydroxyquinoline alanine translation system and application thereof
  • 8-hydroxyquinoline alanine translation system and application thereof
  • 8-hydroxyquinoline alanine translation system and application thereof

Examples

Experimental program
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Embodiment 1

[0111] Embodiment 1: the biocatalytic synthesis of 8-hydroxyquinoline alanine (HqAla) ( Figure 1-Figure 4 )

[0112] The present invention adopts the method of biological enzyme catalysis, utilizes tyrosine phenol lyase (TPL) cloned from Citrobacter freundii (ATCC8090, purchased from American Type Culture Collection (ATCC)) to catalyze 8-hydroxyquinoline Synthesis of 8-hydroxyquinoline alanine, catalytic reaction formula see figure 1 .

[0113] However, the experimental results show that the wild-type tyrosine phenol lyase (TPL, amino acid sequence is SEQ ID NO: 11) cannot catalyze the production of 8-hydroxyquinoline alanine. Therefore, the present inventor analyzed the crystal structure diagram of TPL, selected 448-position phenylalanine, 36-position phenylalanine and 288-position methionine, and introduced NNK mutation (N=A+T+C+G; K=T+G), construct a pEt-TPL mutant library to carry out directed evolution of TPL. 1024 single clones were selected from the mutant library,...

Embodiment 2

[0117] Example 2: Evolution of HqAla-specific aminoacyl-tRNA synthetases

[0118] For site-specific insertion of HqAla 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

[0122] Example 3: Expression of HqAla-green fluorescent protein and identification by mass spectrometry

[0123] The orthogonal tRNA (SEQ ID NO: 1) and the screened nucleotide sequence encoding HqAlaRS (SEQ ID NO: 3) were constructed on the pEVOL vector (purchased from PeterG. The nucleotide sequence (66TAG) (SEQ ID NO: 6) of the easily folded green fluorescent protein was constructed on the pET22b vector (purchased from Novagen), and then co-transformed into Escherichia coli BL21 competent cells (purchased from Quanshijin Company) middle. Pick a single clone and culture it at 37°C for 12 hours in 5ml LB medium containing 50μg / mL ampicillin and 30μg / mL chloramphenicol. Then 1ml of the above culture solution was expanded in 100ml LB medium containing the above antibiotics to OD 600 When it was approximately equal to 1.1, 1 mM HqAla, 1 mM IPTG and 0.2% arabinose (purchased from sigma company) were added to the LB medium to culture the cells, and the control did not add HqAla. ...

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Abstract

The invention relates to an aminoacyl-tRNA synthetase mutant which has an amino acid sequence selected from a group comprising an amino acid sequence as shown in SEQ ID NO:4 and conservative variants of the amino acid sequence as shown in SEQ ID NO:4, wherein the conservative variants have the same enzymatic activity as the amino acid sequence as shown in SEQ ID NO:4. The The invention also provides a translation system which comprises: (i) 8-hydroxyquinoline alanine or its structure analogs; (ii) an orthogonal aminoacyl-tRNA synthetase of the invention; (iii) orthogonal tRNA, wherein the orthogonal aminoacyl-tRNA synthetase realizes prior aminoacylation of the orthogonal tRNA by the 8-hydroxyquinoline alanine or its structure analogs; and (iv) nucleic acid coding target protein, wherein the nucleic acid contains at least one selection codon specifically recognized by the orthogonal tRNA. Finally, the invention provides a tyrosine phenol lyase mutant which can catalyze 8-hydroxyquinoline to generate 8-hydroxyquinoline alanine, and has an amino acid sequence as shown in SEQ ID NO:5.

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 have the same enzymatic activity as the amino acid sequence shown in SEQ ID NO:4. The present invention also relates to a translation system of 2-amino-3-(8-hydroxyquinolin-5-yl) propionic acid (abbreviated as 8-hydroxyquinoline alanine, abbreviated as HqAla). More specifically, the present invention relates to a translation system for site-specifically inserting 8-hydroxyquinoline alanine or its structural analogues into a target protein using the pairing of an orthogonal tRNA and an orthogonal aminoacyl-tRNA synthetase, and using said A method for site-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12P21/02C12N1/21C12N9/88C12N15/60C12P17/12C12R1/19
CPCC12N9/88C12N9/93C12N15/70C12P17/12C12Y401/99002C12Y601/01
Inventor 王江云刘晓红李家松胡诚周庆张维胡美荣周娟作江欢欢
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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