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Acrylyl lysine translation system and application thereof

A technology of acryloyl lysine and acryloyl lysine aminoacyl, which is applied in the field of biochemistry and can solve the problem that inducibility is not widely understood.

Active Publication Date: 2014-01-22
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The importance of good bioorthogonality and fast reaction rates is well recognized in the field, but the importance of inducibility is not widely understood

Method used

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  • Acrylyl lysine translation system and application thereof
  • Acrylyl lysine translation system and application thereof
  • Acrylyl lysine translation system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1: chemical synthesis

[0046] 1. Synthesis of acryloyllysine (AcrK) ( figure 1 ): Add compound N-α-Boc-lysine (2.46g, 10.0mmol, purchased from Shanghai Gil Biochemical Company) and anhydrous sodium carbonate (2.12g, 20.0mmol), and then add 100mL of ethyl acetate and water mixed solvent (ethyl acetate: water (v / v) = 1: 1), slowly add 1.1 equivalents of acryloyl chloride (purchased from TCI company) under stirring in an ice bath ethyl acetate solution. React overnight, adjust the pH to 3 with glacial acetic acid, extract with ethyl acetate, add 100 mL of ethyl acetate hydrogen chloride solution under ice bath after rotary evaporation, stir overnight, a large amount of white solid precipitates, filter, and wash the solid several times with ethyl acetate , the target compound acryloyllysine (1.44 g, 7.2 mmol) was obtained after drying, and the yield was 72%. 1 H NMR (600MHz, D 2 O) δ=1.38-1.47 (m, 2H), 1.55-1.59 (m, 2H), 1.89-1.97 (m, 2H), 3.26 (dd, J 1 =6....

Embodiment 2

[0051] Example 2: Expression of AcrK-green fluorescent protein (GFP) and identification by mass spectrometry

[0052] In order to site-specifically incorporate acryloyllysine (AcrK) into the gene, it is necessary to introduce an acryloyllysine aminoacyl tRNA synthetase / tRNA orthogonal pair into the E.coli host cell used, this orthogonal pair Amber suppressor tRNA (Mb tRNA) from Methanosarcina barkeri CUA Pyl ) / aminoacyl tRNA synthetase (Mb PylRS) pair. Orthogonal tRNA (SEQ ID NO: 1) and acryloyllysine aminoacyl tRNA synthetase mutant (SEQ ID NO: 2) (Heinz Neumann, Sew Y Peak-Chew, Jason W Chin, Genetically encoding Ne-acetyllysine in recombinant proteins, Nature chemical biology, 4, 4, 2008; Neumann H, Hancock SM, Buning R, et al, Mol Cell, 2009 Oct9; 36(1): 153-63) were respectively constructed into the pEVOL vector (American scripps research Peter G. Schultz Laboratory), and then co-transformed into DH10B cells (purchased from Quanshijin Company) containing pEt22b-GFP-151...

Embodiment 3

[0054] Example 3: Expressing AcrK-Ftsz and performing in vivo and in vitro photoclick reactions

[0055] The wild-type Ftsz (SEQ ID NO: 6, derived from Escherichia coli BL21) was constructed on the pEt22b vector (purchased from Novagen), and the orthogonal tRNA (SEQ ID NO: 1) and acryloyllysine aminoacyl tRNA The nucleotide sequences (SEQ ID NO: 3) of the synthetase mutants were respectively constructed on the pEVOL vector (gifted by the Peter G. Schultz laboratory of Scripps Research Institute, USA). A TAG (SEQ ID NO: 8, Ftsz-3TAG, wherein the amino acid at the 3rd position is acryloyl lysine, represented by ) was introduced at the 3rd position of Ftsz by PCR method. Cotransform pEVOL-tRNA (that is, a recombinant vector comprising an orthogonal tRNA sequence (SEQ ID NO: 1)), pEVOL-AcrKRS (that is, a nucleotide sequence comprising an acryloyllysine aminoacyl tRNA synthetase mutant (SEQ ID NO: 1) ID NO: 3) recombinant vector) and pEt22b-Ftsz-3TAG into BL21 (DE3) cells (purchas...

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Abstract

The invention provides an acrylyl lysine translation system which dopes acrylyl lysine into target protein by using an orthogonal tRNA, an orthogonal acrylyl lysine aminoacyl tRNA synthetase and their combination, and also provides a method for specifically doping acrylyl lysine into target protein at a fixed point by using the translation system. The acrylyl lysine translation system comprises: (i) acrylyl lysine; (ii) the orthogonal acrylyl lysine aminoacyl tRNA synthetase; (iii) the orthogonal tRNA, wherein the orthogonal acrylyl lysine aminoacyl tRNA synthetase is used for preferential aminoacylation of the orthogonal tRNA by the acrylyl lysine; and (iv) nucleic acid coding the target protein, wherein the nucleic acid contains at least one selection codon specifically recognized by the orthogonal tRNA. The invention also relates to an application of the mutant protein doped with the acrylyl lysine.

Description

technical field [0001] The invention belongs to the field of biochemistry. Specifically, the present invention provides an acryloyllysine 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 an acryllysine (or AcrK) translation system. More specifically, the present invention relates to an acryloyllysine translation system utilizing orthogonal tRNAs, orthogonal acryloyllysine aminoacyl tRNA synthetases, and their pairings to incorporate acryloyllysine into target proteins, and utilizing the A genetic method for the site-specific incorporation of acryloyllysine in target proteins by the translation system described above. The present invention also relates to the mutant protein containing acryloyl lysine produced by this translation system and this genetic method, and the application of the mutant protein containing ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/00C12N1/21C12N5/10C12N15/70C12N15/85G01N33/68A61K49/00C12Q1/02C12R1/01C12R1/19
Inventor 王江云李发慧孙云潘延超
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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