Method for efficient recombinant expression of restriction endonuclease

Abstract

The invention relates to a method for efficient recombinant expression of restriction endonuclease. The method comprises the following steps: (1) inserting restriction endonuclease genes, transforming a carrier, introducing the carrier into escherichia coli, carrying out screening, culturing and sequencing so as to obtain recombinant expression plasmid of the restriction endonuclease, wherein a plasmid vector is a pET-series expression vector which is adjusted and controlled through a lactose operon and a T7 promoter and induced by IPTG; (2) transforming the recombinant expression plasmid of the restriction endonuclease to BL21(DE3)pLysS competent cell, and carrying out screening, so as to obtain a recombinant expression strain of the restriction endonuclease; and (3) carrying out enlarged culture on the recombinant expression strain of the restriction endonuclease, and performing inducible expression of the restriction endonuclease. By virtue of an inhibiting effect of glucose to background expression of an expression system of lactose operon as well as a strict control effect of a BL21(DE3)pLysS strain to the expression system of lactose operon, a step of methylation protection to host DNA is omitted, so that the recombination expression process is greatly simplified, and the method can be applied to recombination expression of multiple restriction enzymes.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular to a method for highly efficient recombinant expression of restriction endonucleases. Background technique [0002] Restriction endonuclease is an important tool enzyme indispensable for contemporary genetic engineering research and the basis of modern molecular biology. Since the first restriction endonuclease was discovered in the late 1960s, more and more restriction endonucleases have been discovered, purified and applied. [0003] The method for producing restriction endonucleases in the prior art is mainly to clone restriction-modified genes into plasmids, and use specific methylases corresponding to restriction endonucleases to prepare restriction endonucleases, relying on high copies of plasmids The high expression of the target protein has been achieved in several cases. The preparation procedure of this method is cumbersome, the yield of restriction endonuclease is low, and ...

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Problems solved by technology

[0003] The method for producing restriction endonucleases in the prior art is mainly to clone restriction-modified genes into plasmids, and use specific methylases corresponding to restriction endonucleases to prepare restriction endonucleases, relying on high copies of plasmids The high expression of the target protein has been achieved in several cases. The preparation procedure of this method is cumbersome, the yield of restriction endonuclease is low, and the production cost is high. The obtained methylation-protected strain can only specifically protect the host DNA from a certain restriction. Endonuclease cleavage, narrow application range

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