Production method of recombinant NotI restriction endonuclease in escherichia coli

Abstract

The invention discloses a production method of recombinant NotI restriction endonuclease in escherichia coli. The production method comprises the following steps of a) construction of a pCreat Duet-NotI-methylase expression plasmid, b) induction expression and identification of NotI restriction endonuclease and methylase protein, and c) purification and identification of the NotI restriction endonuclease and the methylase protein. According to the production method, an escherichia coli expression system is applied, a coexpression vector is designed for coexpression with a methylase gene, and methylase can protect an escherichia coli genome against an influence of NotI, so that expression of the recombinant NotI in the escherichia coli is realized; purification is performed on NotI protein by affinity chromatography and Superdex 200 gel filtration chromatography and a whole purification process needs 5-6h, so that a simple two-step purification manner greatly shortens a purification procedure and the purification time of the NotI protein, and the purity, a yield and the enzyme activity of the NotI protein are improved and increased; and a novel method of a purification technology of the NotI protein lays a foundation for researching, developing and producing other II-type restriction endonuclease.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for producing recombinant NotI restriction endonuclease in Escherichia coli. Background technique [0002] Restriction endonuclease is a type of enzyme that can recognize a specific nucleotide sequence and cut the phosphodiester bond between two nucleotides at a specific position in each chain, referred to as restriction enzyme. According to the structure of the restriction enzyme, the required cleavage site and the mode of action of the cofactor, the restriction enzyme can be divided into three types, namely the first type (Type I), the second type (Type II) and the third type (Type III ). [0003] The first type of restriction enzyme has the function of modifying and recognizing cleavage at the same time; it also has the ability to recognize a specific base sequence on DNA, and its cleavage site is usually thousands of bases away from the recognition site. For example: E...

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Problems solved by technology

[0004] The second type of restriction enzyme only has the function of recognizing and cutting, and the modification is carried out by other enzymes

[0016] At present, the price of commercialized NotI protein remains high, mainly due to the existence of problems such as low expression, complicated purification, low yield, and low enzyme activity. The existing NotI expression technology is backward, and it is difficult to achieve recombinant NotI in the large intestine. Bacillus; the current NotI purification process in the literature involves anion exchange column, heparin agarose affinity chromatography column, DEAE-agarose column, PEI column and other column elution processes. The entire purification process takes about 3 to 4 days. The process is cumbersome and the protein loss is large