Method for preparing enhanced monomeric bacterial luciferase luxAB

Abstract

The invention discloses a method for preparing an enhanced monomeric bacterial luciferase luxAB. The method comprises the following steps: obtaining a target gene; constructing a recombinant expression vector; obtaining an expression strain containing a recombinant expression plasmid; inducing the expression of a target protein, and purifying the protein; and detecting the activity of the purified enhanced monomeric bacterial luciferase. The unique advantages of fusion type luciferase are utilized to fuse luxA and luxB heterodimer luciferase gene, nine fluorescence intensity enhanced mutants are obtained by screening through error prone PCR mutation and chemical random mutation, a shuffling technology is carried out to obtain enhanced monomeric bacterial luciferase gene luxAB, the enhanced monomeric bacterial luciferase gene luxAB is cloned into an expression vector pET28a, the obtained vector is transformed into a BL21 (DE3) strain to express, a mild breaking technology is used to lyse cells, and an expressed product is purified to obtain the enhanced monomeric bacterial luciferase. The method has the advantages of simple process, short growth cycle, low cost, easy purification, small enzyme activity loss, stable performances of the above enzyme, and important actual application values.

Description

technical field [0001] The invention belongs to the field of bacterial luciferase preparation, in particular to a method for preparing enhanced monomeric bacterial luciferase luxAB. Background technique [0002] Due to their unique physiological characteristics, luminescent bacteria are used as indicators for the determination of toxicants in the environment in environmental monitoring, and luciferase plays a vital role in the process of bacterial luminescence. [0003] Luciferase (English name: Luciferase) is a general term for a class of enzymes that catalyze the oxidation and luminescence of luciferin or aliphatic aldehydes in organisms. [0004] Luciferase has many advantages such as high sensitivity, good specificity, rapid response, easy operation, wide application, and non-toxic effect on organisms. Luciferase is more and more used in medicine, molecular biology, and environmental monitoring. and other related fields. One of them is luciferase in bacteria. In the c...

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Problems solved by technology

[0011] The purpose of the embodiments of the present invention is to provide a method for preparing enhanced monomeric bacterial luciferase luxAB, which aims to solve the problem that the preparation of bacterial luciferase in the prior art is restricted by region and season, and the breeding cycle is long and the breeding cost is high. The post-treatment also needs to go through multiple chemical extraction steps such as centrifugal separation, thiamine fractionation, and column chromatography purification. The process is cumbersome and the application area is relatively narrow.

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