蛇毒类凝血酶安克洛在毕赤酵母中的表达  

Expression of Ancrod,a Snake Venom Thrombin-Like Enzyme,in Pichia pastoris

作　　者：于学玲[1] 李招发[2] 方宏清[2] 周长林[1] 陈惠鹏[2] 

机构地区：[1]中国药科大学生命科学与技术学院,江苏南京210009 [2]军事医学科学院生物工程研究所,北京100071

出　　处：《生物技术通讯》2007年第3期412-415,共4页Letters in Biotechnology

摘　　要：目的:利用毕赤酵母表达具有生物学活性的蛇毒类凝血酶安克洛。方法:根据已知的天然安克洛的氨基酸序列,结合酵母偏好密码子,设计并合成了安克洛基因序列,将其克隆至表达载体pPIC9中,得到表达质粒pPIC9/Ancrod,电转至毕赤酵母GS115(His-)菌株感受态中,通过表达筛选获得工程菌株。采用5L发酵罐对工程菌进行发酵,表达安克洛。在诱导表达阶段,补加甲醇-山梨醇混合碳源。经疏水柱、肝素亲和柱和阳离子柱三步分离纯化得到重组安克洛,并鉴定其体外生物学活性。结果:重组安克洛表观相对分子质量为43000～48000,其糖基化不均匀,去糖基后蛋白肽链的相对分子质量约29000。重组安克洛被证明具有降解并凝固牛纤维蛋白原的活性,其比活力与天然安克洛相近。结论:采用毕赤酵母表达系统表达并获得有生物学活性的重组安克洛,为大规模生产打下了基础。Objective： To express ancrod, a snake venom thrombin-like enzyme, in methylotrophic yeast Pichia pastoris. Methods： The cDNA encoding ancrod was synthesized with a yeast bias codon and inserted into the eukaryotic expression vector pPICg. The constructed plasmid pPICg-Ancrod was then transformed into P.pastoris GSll5 （His-） electrocompetent cells. An engineering strain was finally selected out from the pPICg-Ancrod transformants by small scale induction experiments. Recombinant ancrod was produced in 5-liter bioreactor using a sorbitol-methanol feeding strategy and recovered from the fermentation broth through hydrophobic, affinity and ion exchange chromatography, and its biological activity was characterized in vitro. Results： SDS-PAGE analysis revealed that the recombinant ancrod was heterogeneously glycosylated and running at the expected molecular weight of 43-48 kD, which decreased to about 29 kD after deglycosylation with N-glycosidase F. The fibrinogenolytic and zymographic activity of the recombinant ancrod were determined and were found to be similar to that of the native protein. Conclusion： Recombinant ancrod was successfully expressed and purified from Pichia pastoris, which would be able to lay foundation for large scale production.

关 键 词：蛇毒类凝血酶 重组安克洛 毕赤酵母 

分 类 号：Q78[生物学—分子生物学]