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| Production of Restriction Endonuclease Not I Utilizing CpG DNA Methylase M.Sss I Co-expression Vector |
| WANG Pei1,2, CHEN Kai1,2, GAO Song1,2,3 |
1. Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Huaihai Institute of Technology, Lianyungang 222005, China;
2. Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Huaihai Institute of Technology, Lianyungang 222005, China;
3. Jiangsu Marine Resources Development Research Institute, Lianyungang 222005, China |
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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.
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Received: 16 January 2017
Published: 25 August 2017
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