谷氨酰胺转胺酶基因的结构分析及定点突变  

Structural Analysis and Site-Directed Mutagenesis of Transglutaminase Gene

作　　者：代风娇[1] 詹锐[1] 覃桂[1] 万文杰[1] 何冬兰[1] 

机构地区：[1]中南民族大学生命科学学院,湖北武汉430074

出　　处：《化学与生物工程》2015年第6期23-27,共5页Chemistry & Bioengineering

基　　金：湖北省自然科学基金资助项目(2010CDZ046)

摘　　要：根据分离得到的吸水链霉菌的谷氨酰胺转胺酶基因,预测其三维结构,确定其酶活中心,并构建定点突变体。用PCR扩增谷氨酰胺转胺酶基因,通过SWISS-MODEL在线预测三维结构,找到酶活中心,确定突变位点,将361位的组氨酸(CAC)突变成赖氨酸(AAA),将突变后的基因片段连接到pET-22b(+)上得到重组质粒pET-22b(+)-MTG,并转入大肠杆菌BL21(DE3)中进行表达。结果显示,通过对比已有的三维结构模型,找到了酶活中心,并通过重叠延伸PCR(SOE-PCR)技术成功构建了突变体。Based on transglutaminase gene isolated from Streptomyces hygroscopicus,three-dimensional（3D）structure was predicted,the enzyme active center was determinated,site-directed mutant was built.Enzyme active center was found by applying PCR amplification transglutaminase gene and predicting 3Dstructure by SWISS-MODEL online.After identifying mutation site,361 th His（CAC）was turned into Lys（AAA）,and the mutagenesis was inserted into pET-22b（＋）,and the recombinant plasmid pET-22b（＋）-MTG was transformed to E.coli BL21（DE3）.By comparing with the existing 3D model,the enzyme active center was found,and the mutant was built successfully through SOE-PCR.

关 键 词：谷氨酰胺转胺酶 定点突变 酶活中心 重叠延伸PCR(SOE-PCR) 

分 类 号：Q754[生物学—分子生物学]