重组虎纹捕鸟蛛毒素Ⅺ用pMAL-p2X载体的表达和纯化  

Expression and Purification of Recombinant Huwentoxin-Ⅺ by pMAL-p2X Vector

作　　者：刘君梁[1] 刁建波[2] 唐建洲[1] 谢锦云[1] 梁宋平[1] 

机构地区：[1]湖南师范大学生命科学学院,长沙410081 [2]北京大学生命科学学院蛋白质工程及植物基因工程国家重点实验室,北京100871

出　　处：《中国生物化学与分子生物学报》2005年第1期39-44,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金资助项目 (NO .3 0 170 193 )~~

摘　　要：虎纹捕鸟蛛毒素Ⅺ基因通过PCR扩增 ,插入pMAL p2X载体 ,基因 5′端插入凝血酶切割位点 .在大肠杆菌中经IPTG诱导高效分泌表达 .表达产物N端含麦芽糖结合蛋白 ,融合蛋白被分泌到大肠杆菌的胞间质 .经冷渗透休克后 ,透析脱盐 ,用凝血酶切割融合蛋白 ,再经SuperdexTM75分子筛柱层析、高效液相色谱反相C18柱纯化 ,得到重组虎纹捕鸟蛛毒素Ⅺ .质谱分析表明 ,rHWTX Ⅺ系正确表达产物 ,重组HWTX Ⅺ表现出与天然HWTX Ⅺ一致的生物学活性 .The gene of huwentoxin Ⅺ (HWTX Ⅺ) was amplified by polymerase chain reaction. The amplified gene was cloned into a prokaryotic vector pMAL p2X. At the 5' terminus of the gene, a thrombin cleavage site was inserted. The fusion protein, whose N terminus encoded the maltose binding protein, was expressed in E.coli under IPTG induction. The yield of the fusion protein was 25 mg/L. After cold osmotic shock, the fusion protein was cleaved with thrombin,and the rHWTX Ⅺ was purified by size exclusion chromatography and reversed phase HPLC to a homogeneity. The purified rHWTX Ⅺ, whose yield was 0.8 mg/L, was proved to be correctly expressed by MALDI TOF mass spectrometry. The rHWTX Ⅺ, as a trypsin inhibitor, was analyzed using photospectrometry to have the same biological activity as the native one.

关 键 词：虎纹捕鸟蛛毒素Ⅺ pMAL—p2X 凝血酶 胰蛋白酶抑制剂 

分 类 号：Q966[生物学—昆虫学] Q78