死亡素与泛素在大肠杆菌中的高效融合表达  

High-level fusion expression of thanatin with an ubiqutin fusion partner in Escherichia coli

作　　者：金丰良[1] 张呈文[1] 许小霞[1] 孙强[1] 任顺祥[1] 

机构地区：[1]华南农业大学资源环境学院昆虫学系/教育部生物防治工程研究中心,广州510642

出　　处：《昆虫学报》2009年第5期495-501,共7页Acta Entomologica Sinica

基　　金：国家重点基础研究发展规划("973"计划)项目(2006CB102005);国家青年科学基金项目(30800718);公益性行业科研专项经费(200803005)

摘　　要：死亡素是由21个氨基酸残基组成的广谱抗菌肽。为了高效表达可溶性的死亡素,本研究利用递归式PCR(recursive PCR,rPCR)扩增了死亡素基因thanatin,并将其和家蝇Musca domestica泛素基因ubiquitin构成嵌合基因,克隆到表达载体pET-32a,再与硫氧还蛋白融合后构建表达载体pET-TRX-UBI-THA。将酶切和测序鉴定正确的质粒转化表达宿主菌BL21,经0.6mmol/L IPTG诱导,TRX-UBI-THA融合蛋白得到了高效可溶性表达。SDS-PAGE和Western blot检测结果表明融合蛋白的分子量为28.9kD,与预期的结果一致,表达量占菌体总蛋白的46%。Western blot分析结果显示融合蛋白能与Ni-NTA鏊合物特异性的结合,表明在融合蛋白的N-端带有6×His标签。利用C-端带有6×His标签的泛素C-端水解酶对融合蛋白进行切割,切割产物经Ni2+-NTA亲和柱和HPLC纯化(纯化量为5.4mg/L),Tricince-SDS-PAGE电泳得到单一的泛素蛋白条带。电喷雾质谱(ESI-MS)分析表明,纯化的泛素分子量为2.57kD,与通过氨基酸预测的分子量完全一致。利用琼脂孔穴扩散法对泛素活性进行检测,结果显示纯化的泛素对大肠杆菌K12D31和金黄色葡萄球菌Staphylococcus aureus具有较强的活性抑制。本研究表明,利用泛素融合技术可以高效表达可溶性的死亡素。Thanatin is one of the antibacterial peptides which consists of 21 amino acids and has a broad spectrum of antimicrobial activities. To explore a cost-effective approach for high-level expression of the thanatin in Escherichia coli, the cDNA fragment encoding thanatin with preferred codons of E. coli was obtained by recursive PCR （rPCR） and fused to the C-terminal of ubiquitin （UBI） from housefly, Musca domestica. The fused gene was inserted into the plasmid pET32a to construct the expression vector pET- TRX-UBI-THA and express the fusion protein with 6 × His tag in E. coli BL21. The fusion protein was expressed in soluble form under the optimized conditions at a high-level （more than 46% of the total proteins）. With 6 × His tag, the proteins were easily purified by Ni^2＋ -NTA chromatography. The purified proteins were efficiently cleaved by ubiquitin carboxyl-terminal hydrolase which linked with 6 × His tag at the carboxyl-terminus （ UCH-6 × His）, yielding recombinant thanatin with significant antimicrobial activity. After contaminants removal by Ni^2＋ -NTA chromatography, recombinant thanatin was purified by reversed phase HPLC, 5.4 mg of pure active thanatin was obtained from 1 L culture medium. ESI-MS analysis showed that the molecular weight of the purified recombinant thanatin was 2. 57 kD, which perfectly matched the mass calculated from the amino acid sequence. The antimicrobial assay showed that the purified thanatin has high activities against E. coli Kl2 D31 and Staphylococcus aureus. Our results demonstrated that functional thanatin can be produced in sufficient quantity using the ubiquitin fusion technique at a low cost.

关 键 词：抗菌肽 死亡素 泛素 融合表达 纯化 活性检测 

分 类 号：Q78[生物学—分子生物学] S43[农业科学—农业昆虫与害虫防治]