Expression and applications of novel non-restriction endonuclease in escherichia coli

Abstract

The invention utilizes the property that nonspecific endonuclease from Anabaena sp. is provided with natural inhibitor protein to change the original expression way of the nonspecific endonuclease, i.e. to change secretory expression into endoenzyme high efficient expression. The invention adopts a strong promoter to largely express in cells in a way of inclusion bodies with no activity; wherein, the moment when the endonuclease is expressed, the natural inhibitor protein is expressed together so as to eliminate the strong toxicity that farthing activated nuclease imposes on the cells; protein engineering technology is used for replacing a non-activated central amino acid, thus reducing the possibility of forming multimer in renaturation, improving efficiency, and furthermore, raising the output in terms of improving the expression efficiency and the renaturation efficiency.

Description

technical field The invention relates to a method for producing a non-specific endonuclease by recombinant DNA technology. The enzyme can degrade various forms of nucleic acids (including DNA and RNA), and can be used to remove nucleic acids in biological materials containing nucleic acids as impurities or eliminate the influence caused by nucleic acids. Background technique Non-specific endonucleases are a class of hydrolytic enzymes that can degrade various forms of DNA and RNA, and have high phosphodiester bonds for single-stranded, double-stranded, linear, circular and supercoiled forms of DNA and RNA. activity to produce 5'-phosphate nucleotides or 5'-phosphate oligonucleotides, and has no sequence requirement for nucleic acids. It can be used to remove nucleic acids in biological materials or to exclude interference caused by nucleic acids. A typical representative of this type of enzyme is a non-specific endonuclease derived from Serratia marcescens, which has been...

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Problems solved by technology

Therefore, the yield of this enzyme is low, the process is cumbersome, and the price is relatively expensive, which is not suitable for use in my country.

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