应用不同载体探索环二肽合成酶AlbC的高效可溶性表达  

Efficient Soluble Expression of Cyclic Dipeptide Synthetase AlbC Based on Different Vectors

作　　者：石雄飞 袁思棋 郑佳 王洪 李玲珊 刘君 SHI Xiongfei;YUAN Siqi;ZHENG Jia;WANG Hong;LI Lingshan;LIU Jun(School of Bioengineering,Sichuan University of Science and Engineering,Yibin,Sichuan 644000;Wuliangye Group Co.Ltd.,Yibin,Sichuan 644000,China)

机构地区：[1]四川轻化工大学生物工程学院,四川宜宾644000 [2]宜宾五粮液集团有限公司,四川宜宾644000

出　　处：《酿酒科技》2023年第7期65-71,共7页Liquor-Making Science & Technology

基　　金：五粮液校企合作项目(CXY2020ZR02);四川省科技厅重点研发项目(No.2021YFS0341);四川理工学院人才项目(2017RCL25)。

摘　　要：AlbC是一种环二肽合成酶,可用于合成具有抗菌性和抗肿瘤性的环二肽及后续衍生物。近年来,AlbC的产物在白酒及其副产物黄水中被相继检出,对赋予白酒健康功能起着至关重要的作用。为实现albC基因的高效可溶性表达,本研究以实验室保藏的枯草芽孢杆菌Y1为模板克隆出albC基因,构建了pQE30-albC,pET28a-albC,pET28a-SUMO-albC,pGEX-6P-1-albC 4个重组质粒,并探究了不同诱导温度下每个重组质粒的诱导表达情况。结果表明:(1)pQE30-albC不表达或者表达量低;pET28a-albC正确表达,但表达量和可溶性表达量都较低,纯化后融合蛋白浓度为1.0 mg/L;pET28a-SUMO-albC正确表达,且实现可溶性表达,纯化后融合蛋白浓度为30 mg/L,切除SUMO标签蛋白后浓度为22 mg/L;pGEX-6P-1-albC正确表达,且实现可溶性表达,纯化后融合蛋白浓度为44 mg/L,切除GST标签蛋白后浓度为34 mg/L;(2)拥有GST促溶标签的pGEX-6P-1载体是4组载体中达到albC基因可溶性表达水平最高的载体;(3)在4种载体中,albC基因最佳可溶性表达条件为:以pGEX-6P-1为载体,在20℃的诱导温度下诱导表达18 h;可成功实现albC基因的高效可溶性表达。AlbC is a cyclic dipeptide synthetase that can be used to synthesize cyclic dipeptide and subsequent derivatives rich in antibacterial and anti-neoplastic properties.In recent years,AlbC products have been detected in Baijiu and its byproduct yellow water,which plays an important role in endowing Baijiu with healthcare function.In order to achieve efficient soluble expression of albC gene,we cloned albC gene by using Bacillus subtilis Y1 stored in our laboratory as the template,and constructed four recombinant plasmids:pQE30-albC,pET28a-albC,pET28a-SUMO-albC,and pGEX-6P-1-albC.The induced expression of each recombinant plasmid at different induction temperatures was investigated.The results showed that:(1)pQE30-albC was not expressed or the expression level was low;pET28a-albC was correctly expressed,but the expression level and soluble expression level were low,and the fusion protein concentration was 1.0 mg/L after purification;pET28a-SUMO-albC was correctly expressed and soluble,the fusion protein concentration was 30 mg/L after purification,and 22 mg/L after excising SUMO tag protein;pGEX-6P-1-albC was correctly expressed and soluble,the fusion protein concentration was 44 mg/L after purification,and 34 mg/L after excising SUMO tag protein.(2)The pGEX-6P-1 vector with GST tag was the vector with the highest soluble expression level of albC gene among the four vectors.(3)The optimal soluble expression conditions of albC gene were as follows:pGEX-6P-1 as the vector,inducing at 20℃for 18 h.Finally,the efficient soluble expression of albC gene was achieved.

关 键 词：环二肽合成酶AlbC 载体 可溶性表达 

分 类 号：TS262.3[轻工技术与工程—发酵工程] TS261.7[轻工技术与工程—食品科学与工程]