AcAPc2自剪切融合蛋白表达载体pTWIN1-AcAPc2的构建和表达

Construction and Expression of Protein Self-splicing Prokaryotic Expression Vector pTWIN1-AcAPc2

为表达和获取具有抗凝血功能的犬钩虫抗凝血肽(A cAP c2),采用搭桥PCR方法合成犬钩虫抗凝血肽全长双链cDNA序列,A cAP c2 cDNA全序列连入表达载体pTW IN 1上构建成具有蛋白自剪切功能的表达载体pTW IN 1-A cAP c2,将阳性重组子转入表达型大肠杆菌E.coli ER 2566进行表达。表达的融合蛋白A cAP c2-in te in2-CBD为可溶性蛋白,且融合蛋白约占菌体总蛋白的30.1%,经蛋白质印迹分析确定表达产物是具有CBD蛋白的特异性融合蛋白。融合蛋白A cAP c2-in te in2-CBD经几丁质柱高效亲和纯化,并经β-巯基乙醇诱导的独特的在柱自剪切后,得到目的蛋白A cAP c2,经SDS-PAGE分析在21 KD处呈现目的条带,所得的可溶性A cAP c2分子量符合其天然活性的二聚体形式。对A cAP c2表达和纯化工艺上的改进,方便了A cAP c2的获取,A cAP c2氨基酸序列的生物信息学分析初步阐明了A cAP c2的结构和功能的关系,为进一步研究A cAP c2的抗凝血机制及其作为抗凝血药的临床应用奠定了基础。

To express recombinant Ancylostoma caninum anticoagulant peptide-c2 （AcAPc2）, a whole cDNA fragment encoding AcAPc2 was achieved by ligation- PCR and inserted into prokaryotic expression vector pTWIN1 for constructing the specific self-splicing prokaryotic expression vector, pTWIN1-AcAPc21 positive recombinants were transformed into E. coli ER2566 for expression research. The recombinant protein, AcAPc2-intein2-CBD, was soluble and expressed in E. coli ER2566 （about 30. 1% fusion protein in total protein）. AcAPc2-intein2-CBD was characterized to be 41 KD by SDS-PAGE and identified by Western-blot. The recombinant fusion protein was purified to a efficiently high degree by chitin affinity chromatography. After the process of specific self-splicing induced by β-Mercaptoethanol, the target protein, AcAPc2, was obtained, characterized to be 21 KD by SDS PAGE and migrated as a dimmer. Molecular weight of AcAPc2 conformed to native dimmer. Bio-information analysis indicated relationship between secondary construction of AcAPc2 and biologic function. These findings greatly facilitate the purification of AcAPc2 and are very important for the additional studies on its anti-coagulation mechanism and its clinical application as anti-coagulation medicine.