nε-(1-methylcycloprop-2-enamide)-lysine translation system and its application

A translation system and methyl ring technology, applied in enzymes, cells modified by introducing foreign genetic material, biochemical equipment and methods, etc., can solve problems affecting the conformation of proteins themselves
CN103667202BActive Publication Date: 2016-01-13INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
Publication Date
2016-01-13

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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.
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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 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 mutant p...

Claims

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