Production of Restriction Endonuclease Not Ⅰ Utilizing CpG DNA Methylase M.Sss Ⅰ Co-expression Vector
10.13523/j.cb.20170808
- VernacularTitle:利用CpG DNA甲基化酶M.Sss Ⅰ共表达载体制备限制性内切酶Not Ⅰ
- Author:
Pei WANG
;
Kai CHEN
;
Song GAO
- Keywords:
Recombinant protein expression;
Restriction endonuclease;
Methylase
- From:
China Biotechnology
2017;37(8):51-58
- CountryChina
- Language:Chinese
-
Abstract:
Restriction endonucleases are important molecular biology tools for DNA recombination.Because of the cleavage of DNA,their recombinant expression is difficult with low yields and complicated purification processes.In commercial productions,the technology that uses specific methylases to protect host DNA from digestion of the expressed restriction enzymes was cumbersome and practically limited.For solving this problem the expression of restriction enzyme Not Ⅰ was performed by using the DNA methylase M.Sss Ⅰ derived from Spiroplasma sp.MQ1 which specifically kept CpG sequence methylated.The methylated DNA was protected from the cutting of Not Ⅰ whose recognition sequence contained CpG.The gene of methylase M.Sss Ⅰ was introduced into Escherichia coli ER2566 and constitutively expressed,resulting in the CpG methylation pattern of the host DNA.Restriction enzyme Not Ⅰ was successfully expressed in this E.coli strain.Furthermore,by adding a purification tag to one terminus of the enzyme,recombinant Not Ⅰ was prepared as a highly purified and active product through two simple Ni-affinity and anion exchange chromatography steps.This expression system can be applied for the preparation of a series of restriction enzymes with CpG in their recognition sequences.
