蛇毒类凝血酶calobin在毕赤酵母中的表达  

Expression of snake venom thrombin-like enzyme calobin in Pichia pastoris

作　　者：袁盛凌[1] 王芃[1] 陶好霞[1] 展德文[1] 王艳春[1] 王令春[1] 刘纯杰[1] 张兆山[1] 

机构地区：[1]中国人民解放军军事医学科学院生物工程研究所,北京100071

出　　处：《生物工程学报》2009年第4期526-532,共7页Chinese Journal of Biotechnology

摘　　要：蛇毒类凝血酶是临床上防治血栓栓塞性疾病的有效药物。参照朝鲜蝮蛇(Agkistrodon caliginosus,Korean Viper)类凝血酶calobin基因序列(GenBank AccessionNo.U32937.1),将人工合成的calobin基因克隆到酵母表达载体pPICZαA,于毕赤酵母中表达,得到了分子量约为32kD的重组calobin蛋白,经甲醇诱导培养,表达产物可获得3.5g/L的高表达量。重组蛋白经过阴离子交换柱Q-Sepharose Fast Flow和分子筛Sephacryl-S-100凝胶过滤层析等纯化步骤进行了初步纯化。纯化后的重组calobin可以在纤维蛋白原平板上形成水解圈,经SDS-PAGE实验显示,重组蛋白能水解纤维蛋白原的Aα链,产生一条约40kD左右的降解带。在实验中未能发现重组calobin对纤维蛋白原的凝固作用。Thrombin-like enzymes （TLEs） are studied widely because of their therapeutic potential in myocardial infarction and thrombotic diseases. We synthesized the DNA fragment encoding thrombin-like enzyme calobin from Agkistrodon caliginosus （Korean Viper） venom by fusion PCR and expressed it in Pichia pastoris. After induction by 0.5% methanol for 48 h, the expression level of recombinant calobin reached 3.5 g/L in medium. The recombinant calobin was purified by Q-Sepharose Fast Flow ion-exchange chromatography and Sephacryl-S-100 gel filtration chromatography. Purified sample had a molecular weight of 32 kD shown in SDS-PAGE. It hydrolyzed fibrinogen and formed a light white hydrolysis circle in fibrinogen plate. SDS-PAGE analysis showed that recombinant calobin cleaved Aα-chain of fibrinogen specifically, and produced an appropriately 40 kD new band. However, we failed to find its fibrin-clot formation activity.

关 键 词：蛇毒类凝血酶 毕赤酵母 calobin 纤维蛋白原 

分 类 号：Q78[生物学—分子生物学]