Preparation and preservation method for colon bacillus acetohydroxyacid synthase

Abstract

The invention provides a method for preparing AHAS (Acetohydroxyacid Synthase) which has good purity and high activity and can exist stably. The method comprises the following steps: upstream and downstream primers are designed according to a gene sequence of a colon bacillus AHAS catalytic subunit, wherein the designed upstream primer carries an Xho I enzyme digestion site and the downstream primer carries a BamHI enzyme digestion site; a colon bacillus BL21 strain genomic DNA (Deoxyribonucleic Acid) is used as a template and a target segment ahas is obtained by a PCR (Polymerase Chain Reaction) amplification technology; the target segment ahas is connected onto an expression carrier PGEX-AT-1 to obtain a recombinant plasmid PGEX-4T-1-ahas; inducible expression is realized in a prokaryotic expression system; an expressed enzyme is purified by agarose gel resin modified by glutathione sulfur transferase (GST) so as to obtain the acetohydroxyacid synthase with good activity.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to methods for cloning, soluble expression, purification and preservation of Escherichia coli acetolactate synthase gene. Background technique [0002] Acetolactate synthase (acetolactate synthase, AHAS, EC4.1.3.18) was first discovered in Escherichia coli (E.coli). So far, AHAS genes derived from 60 different biological species can be searched in GenBank, and these organisms are mainly bacteria, algae, fungi, etc. Usually, there may be many genes with high homology with AHAS gene in an organism, but not every gene can play a corresponding role with AHAS, and the AHAS gene in different parts of the same organism There are also large differences in copy number. In some lower organisms, the nucleotide sequence of AHAS is highly conserved, and its similarity is generally about 80%. The AHAS of Escherichia coli is a tetramer consisting of two large subunits and two smal...

AI Technical Summary

Problems solved by technology

The method of preparing recombinant AHAS by genetic engineering technology has also been reported. Most of the vectors used are pET series vectors, but the amount of expressed AHAS protein is relatively low, or it is expressed in the form of inclusion bodies, and it needs to go through steps such as denaturation and renaturation. The operation is complicated and the cost is high, and these methods have not solved the problem of poor stability and easy inactivation of the enzyme, so there is no commercial AHAS product sales at home and abroad

The existence of these problems seriously hinders the in-depth and comprehensive research on AHAS and the search for new AHAS inhibitors, and also largely limits its wide application in many important industrial fields.

Images