Method for efficiently expressing extracellular Serratia marcescens non-specific nuclease based on Bacillus subtilis expression system

Abstract

The invention discloses a method for efficiently expressing extracellular Serratia marcescens non-specific nuclease based on a Bacillus subtilis expression system. The method is characterized by comprising the following steps: taking Bacillus subtilis as a host expression system, and utilizing the extracellular secretion capacity of the Bacillus subtilis to realize the high-efficiency expression of the extracellular Serratia marcescens non-specific nuclease. The specific activity of the extracellular Serratia marcescens non-specific nuclease recombined and expressed by the invention is 3 timesas high as that of current commercial products. The Bacillus subtilis secretion expression system selected by the invention fundamentally overcomes the defects that endotoxin introduced by the self characteristics of other expression systems and the glycosylation modification affect enzyme activity or limit the application of the system, efficient secretion-type expression does not need to breakcells, the purification steps are simpler and more convenient, linear amplification can be realized, scale production is convenient, and the method is worthy of popularization.

Description

technical field [0001] The invention relates to the fields of biochemistry and molecular biology, in particular to a method for efficiently expressing non-specific nucleases of prodigies based on a bacillus expression system. Background technique [0002] Non-specific nucleases are a class of highly active hydrolytic enzymes, whose greatest feature is that they can non-specifically degrade almost all types of nucleic acids, and have no strict preference for nucleic acid sequences. Among them, non-specific nucleases (extracellular Serratia marcescens nuclease, SMNE), is a sugar non-specific endonuclease secreted extracellularly, which has low requirements for substrate specificity and can non-specifically degrade all forms of nucleic acids into 2-4 nucleotides. Therefore, its most typical use is mainly the removal of nucleic acids in the upstream process of recombinant protein engineering purification, reducing the viscosity of samples to facilitate subsequent processing, and...

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Problems solved by technology

[0006] In view of the above-mentioned expression using E. coli as the expression host, the ability of E. coli to preserve and secrete proteins is insufficient, and the growth of the bacteria is inhibited or even died, and the operation steps are cumbersome, which affects the protein production and reduces the activity of the enzyme; When expressing, the post-translational modification of the eukaryotic expression system affects the SMNE activity derived from the prokaryotic, and the specific activity is lower than the original source. The present invention provides a new recombinant expression method to produce and prepare non-specific nucleic acids with high specific activity. Enzymes, the specific scheme is as follows:

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