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

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 Accession No. U32937.1), 将人工合成的calobin基因克隆到酵母表达载体pPICZaA, 于毕赤酵母中表达, 得到了分子量约为32 kD的重组calobin蛋白, 经甲醇诱导培养, 表达产物可获得3.5 g/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Aa链, 产生一条约40 kD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。

Abstract:

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.