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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2017, Vol. 37 Issue (8): 51-58    DOI: 10.13523/j.cb.20170808
技术与方法     
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
王佩1,2, 陈凯1,2, 高嵩1,2,3
1. 淮海工学院江苏省海洋药物活性分子筛选重点实验室 连云港 222005;
2. 淮海工学院江苏省海洋生物产业技术协同创新中心 连云港 222005;
3. 江苏省海洋资源开发研究院 连云港 222005
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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摘要： 限制性核酸内切酶是DNA重组的重要分子生物学工具。由于其本身对DNA有切割作用导致其重组表达在技术上十分困难，产率低、提纯程序复杂。而商业化生产所采用的利用专一型甲基化酶保护宿主DNA的限制酶表达技术流程繁琐、实用性有限。为表达Not I限制酶，采用来源于Spiroplasma sp.MQ1的DNA甲基化酶M.Sss I特异性甲基化CpG序列，甲基化后的DNA会免受识别位点中包含CpG序列的限制酶Not I的切割。将甲基化酶M.Sss I导入大肠杆菌表达宿主ER2566后，M.Sss I基因在宿主中持续表达并甲基化宿主DNA成CpG甲基化样式；利用此表达体系制备限制性内切酶Not I获得成功。并借助引入纯化标签经过简便的Ni亲和层析和阴离子交换层析2步层析洗脱纯化，制备了高活力高纯度的重组限制酶Not I。此表达体系可应用于一系列识别位点中包含CpG序列的限制酶的表达。
关键词： 重组蛋白表达;  甲基化酶;  限制性内切酶    
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.
Key words: Restriction endonuclease    Recombinant protein expression    Methylase
收稿日期: 2017-01-16 出版日期: 2017-08-25
ZTFLH:  Q819  
基金资助: 国家自然科学基金（31470275，31300652）、江苏省自然科学基金（BK20130406）、江苏省高校自然科学基金（13KJB180003）、江苏省高校优势学科建设工程资助项目
通讯作者: 高嵩     E-mail: gaosong@hhit.edu.cn
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引用本文:

王佩, 陈凯, 高嵩. 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[J]. 中国生物工程杂志, 2017, 37(8): 51-58.

WANG Pei, CHEN Kai, GAO Song. Production of Restriction Endonuclease Not I Utilizing CpG DNA Methylase M.Sss I Co-expression Vector. China Biotechnology, 2017, 37(8): 51-58.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170808    或    https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I8/51

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