Selenotyrosine translation system and application thereof

A technology of tyrosine and selenogenesis, applied in the field of biochemistry, can solve problems such as limited application

Active Publication Date: 2019-08-13
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

However, the optimum pH values ​​of wild-type and mutant PTEs are usually greater than 8.5, which may li

Method used

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  • Selenotyrosine translation system and application thereof
  • Selenotyrosine translation system and application thereof
  • Selenotyrosine translation system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Embodiment 1: the chemical synthesis of selenotyrosine (SeHF) ( figure 1 )

[0090] Step 1: Synthesis of diethyl 2-acetamido-2-(4-nitrobenzyl)malonate (compound 2)

[0091] A mixture of 1-(bromomethyl)-4-nitrobenzene (1.0 g, 1 eq), diethyl 2-acetamidomalonate (1.2 g, 1.2 eq) and tBuOK (0.63 g, 1.2 eq) Place in a round bottom flask containing 20 mL of EtOH and monitor reflux by TLC overnight. After the reaction was complete, the mixture was cooled to room temperature, filtered and washed with EtOH. The residue was subjected to flash chromatography on silica gel using ethyl acetate / hexane (1:1 mix) as eluent to afford the title compound (1.23 g, 75.5% yield) as a white solid.

[0092] 1H NMR (500MHz, CDCl3) δ8.12(d, 2H), 7.18(d, 2H), 6.56(s, 1H), 4.28(q, 4H), 3.77(s, 2H), 2.04(s, 3H) , 1.3(t,6H).13C NMR(CDCl3)δ169.40, 167.05, 147.27, 143.15, 130.72, 123.46, 66.86, 63.02, 37.59, 23.02, 14.01.

[0093] Step 2: Synthesis of diethyl 2-acetylamino-2-(4-aminobenzyl)malona...

Embodiment 2

[0103] Example 2: Evolution of SeHF-specific aminoacyl-tRNA synthetases

[0104] In order to site-specifically insert SeHF 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 Tyr CUA) / 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 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-iw1 library, and the construction method of the mutation library is: select 6 sites (Tyr32, Leu65, Phe108, Gln109, Asp158, and Leu162) on the MjYRS gene and introduce NNK mutation ( N=A+T+C+G; K=T+G), the other 6 sites (Ile63, Ala67, His70, Tyr114, Ile159, Val164) were either randomly mutated to G...

Embodiment 3

[0108] Example 3: Expression of SeHF-arPTE and identification by mass spectrometry

[0109] To determine the efficiency and fidelity of SeHF incorporation into proteins, we replaced Tyr309 with an amber stop codon in the C-terminal His6-tagged arPTE. In SeHFRS, MjtRNA Tyr CUA Under the condition of co-existing with 0.5mM SeHF, arPTE was expressed by Escherichia coli, and no SeHF was added as a negative control.

[0110] The specific steps are: construct the orthogonal tRNA (SEQ ID NO: 1) and the screened nucleotide sequence encoding SeHFRS (SEQ ID NO: 3) into the pEVOL vector (purchased from Peter G. ); the nucleotide sequence (SEQ ID NO: 5) encoding arPTE was constructed on the pET-22b vector (purchased from Novagen Company), and was designed by using TransStartFastPfu (purchased from Quanshijin Company) DNA polymerase and primers by PCR The Y309TAG mutation was introduced in arPTE-pET22b. The two plasmids were co-transformed into BL21(DE3) cells (purchased from Quanshiji...

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Abstract

The invention relates to an aminoacyl-tRNA synthase mutant, which is an orthogonal aminoacyl-tRNA synthase. The amino acid sequence contained in the mutant is selected from the amino acid sequence shown in SEQIDNO:4 or its conservative variants or homologues with the same enzyme activity. The invention further provides a selenotyrosine translation system, which comprises (i) selenotyrosine, (ii) the orthogonal aminoacyl-tRNA synthetase, (iii) orthogonal tRNA and (iv) nucleic acid for encoding target protein, wherein the orthogonal aminoacyl-tRNA synthetase adopts the selenotyrosine for aminoacylation of the orthogonal tRNA preferentially, and the nucleic acid contains at least one selection codon for specifically recognizing the orthogonal tRNA. Moreover, the invention provides a method for modifying protease by utilizing the design of the selenotyrosine translation system and protease produced by encoding through 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 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, wherein the conservative variants have the same enzymatic activity as the amino acid sequence shown in SEQ ID NO:4. The invention also relates to a selenotyrosine (abbreviated as SeHF) translation system and its application. [0002] Specifically, the present invention relates to a selenotyrosine translation system for site-specific insertion of selenotyrosine into a target protein by using a pair of orthogonal tRNA and orthogonal aminoacyl-tRNA synthetase, and using the translation system in A method for site-specific insertion of selenotyrosine into target proteins. The present...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/52C12N9/16C12P21/02
CPCC12N9/93C12N9/16C12Y601/01C12P21/02
Inventor 王江云安晓景王天元韩明杰黄爱萍陈超江欢欢
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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