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N<epsilon>-(1-methylcyclopropyl-2-acrylamide)-lysine translation system and application thereof

A methyl ring, enamide technology, applied in the field of biochemistry, can solve problems such as affecting the conformation of the protein itself

Active Publication Date: 2014-03-26
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

Recently, it has been reported that norbornene can perform efficient cycloaddition reactions with macrocyclic tetrazole compounds. However, due to the relatively large size of norbornene, it may affect the conformation of the protein itself. Therefore, we hope that by extending the gene The method of coding incorporates amino acids containing cyclopropene functional groups at the specific sites of proteins, and uses the cyclopropene functional groups to undergo cycloaddition reactions with tetrazole compounds under a certain wavelength of ultraviolet light, thereby performing efficient and controllable initiation. spot-specific protein labeling

Method used

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  • N&lt;epsilon&gt;-(1-methylcyclopropyl-2-acrylamide)-lysine translation system and application thereof
  • N&lt;epsilon&gt;-(1-methylcyclopropyl-2-acrylamide)-lysine translation system and application thereof
  • N&lt;epsilon&gt;-(1-methylcyclopropyl-2-acrylamide)-lysine translation system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1: chemical synthesis

[0046] 1. N ε The chemical synthesis of -(1-methylcycloprop-2-enamide)-lysine (CpK)( figure 1 )

[0047] For the chemical synthesis route of CpK see figure 1 . Concrete synthesis reaction steps are as follows:

[0048] Into a 1000 mL round bottom flask equipped with a magnetic stirrer and a thermometer were added sodium azide (6.5 g, 100 mmol), 2M sodium hydroxide solution (200 mL, 40 mmol), tetrabutylammonium bromide (TBAB) (80 mg, 0.25 mmol ) and 100 mL of hexane (100 mL), stirred vigorously on an ice bath at 0°C. After complete dissolution, trifluoromethanesulfonic anhydride (8.2 mL, 50 mmol) was slowly added dropwise with a syringe, and reacted for 10 minutes. Another ethyl 2-methylacetoacetate (3.53mL, 25mmol) was dissolved in 100mL of acetonitrile, then the ethyl 2-methylacetoacetate solution was added to the reaction flask through a funnel, and then 10mL of acetonitrile was added, and the initial no The colored reaction s...

Embodiment 2

[0063] Example 2: Evolution of CpK-specific aminoacyl-tRNA synthetases

[0064] For the site-specific insertion of CpKs in the gene, an aminoacyl-tRNA synthetase / tRNA orthogonal pair derived from Methanosarcina barkeri needs to be introduced into the E.coli host cell used Amber suppresses lysyl tRNA (Mb tRNA CUA Pyl ) / lysyl tRNA synthetase (Mb PylRS, wild type, its amino acid sequence is SEQ ID NO: 2) pair.

[0065]The Mb PylRS mutant library was constructed in the kanamycin-resistant pBK plasmid (purchased from the Peter G. Schultz laboratory of the Scripps Research Institute in the United States), and located between the promoter and terminator of E. coli glutamine synthetase on the plasmid . The synthetic enzyme mutation library used is the pBk-CpK library, and the construction method of the mutation library is: select 5 sites (L266, L270, Y271, L274 and C313) on the Mb PylRS gene, and perform random mutation by PCR . Positive and negative screens were then performed t...

Embodiment 3

[0067] Example 3: Expressing CpK-myoglobin and performing an in vitro photoclick reaction

[0068] Orthogonal tRNA (SEQ ID NO: 1) and the nucleotide sequence encoding myoglobin (4TAG) (SEQ ID NO: 5) were constructed on the pBAD vector (purchased from the laboratory of Peter G. Schultz, Scripps Research Institute, USA) , the screened nucleotide sequence (SEQ ID NO: 3) encoding CpKRS was constructed on the pBK vector (purchased from Peter G. company). Pick a single clone and grow to OD at 37°C 600 When it was approximately equal to 0.5, 1 mM CpK and 0.2% arabinose (purchased from sigma company) were added to the LB medium to culture the cells, and no CpK was added to the control. After 6-7 hours, the bacteria were harvested, and the protein was purified by Ni-NTA, and analyzed by SDS-PAGE electrophoresis ( Figure 4 a).

[0069] We found that the full-length myoglobin can only be purified in the medium with CpK, which indicates that the screened CpKRS can specifically recogn...

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Abstract

The invention relates to an aminoacyl-tRNA (transfer ribonucleic acid) synthetase mutant containing an amino acid sequence selected from groups consisting of amino acids shown by SEQ ID NO:4 and conservative variants thereof. The invention provides a CpK (short for N<epsilon>-(1-methylcyclopropyl-2-acrylamide)-lysine) translation system for fixed point specific insertion of N<epsilon>-(1-methylcyclopropyl-2-acrylamide)-lysine in a target protein through pairing of orthogonal tRNA and orthogonal aminoacyl-tRNA synthetase, and a method for fixed point specific insertion of CpK in the target protein by using the translation system. The CpK translation system comprises (i) N<epsilon>-(1-methylcyclopropyl-2-acrylamide)-lysine, (ii) the orthogonal aminoacyl-tRNA synthetase, (iii) the orthogonal tRNA and (iv) a nucleic acid for coding the target protein, wherein the orthogonal aminoacyl-tRNA synthetase is used for realizing aminoacylation of the orthogonal tRNA preferentially by using CpK, and the nucleic acid contains at least one selective codon specifically identified by the orthogonal tRNA. The invention also relates to an application of a mutant protein for fixed point specific insertion of CpK in protein labeling through a light click reaction with a tetrazole compound.

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 the amino acids shown in SEQ ID NO: 4 and their conservative variants. The present invention also relates to a kind of N ε -(1-methylcycloprop-2-enamide)-lysine (abbreviated as CpK) translation system. More specifically, the present invention relates to a CpK translation system for site-specifically inserting CpK into a target protein using a pair of orthogonal tRNA and orthogonal aminoacyl-tRNA synthetase, and a method for site-specifically inserting CpK into a target protein using the translation system . The present invention also relates to the CpK-containing mutant proteins produced by this translation system and this method, for example, CpK-inserted myoglobin and enhanced green fluorescent protein mutants, and the use of CpK-inserted muta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N1/21C12N5/10C12P21/00C12P21/02
CPCC07K14/00C07K14/805C12N9/93C12Y601/01006
Inventor 王江云潘延超江欢欢高峰
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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