碳酸酐酶Ⅱ基因克隆及在毕赤酵母中的异源表达  

Cloning of a gene encoding carbonic anhydrase Ⅱ and heterologous expression in Pichia pastoris

作　　者：卫玲[1] 赵莹[1] 徐晓晶[1] 

机构地区：[1]上海生物芯片有限公司/生物芯片上海国家工程研究中心,上海201203

出　　处：《上海医药》2015年第17期74-78,共5页Shanghai Medical & Pharmaceutical Journal

基　　金：国家863项目(2006AA02A252);上海市科委创投联动计划(13CT1900900)

摘　　要：碳酸酐酶Ⅱ(carbonic anhydraseⅡ,CAⅡ)是参与机体多种代谢活动和病理活动的一种重要催化酶。通过基因重组、电转化等方式,得到能异源高表达人CAⅡ的毕赤酵母(Pichia.pastoris)GS115工程菌。对工程菌进行培养及甲醇诱导后,经离子交换层析柱分离纯化后得到较纯的重组CAⅡ。通过Western blotting、酶活力及圆二色谱检测发现重组和商品化CAⅡ具有相似的酶活力和构象,为CAⅡ今后进一步在临床的研究和工业应用打下了基础。Carbonic anhydrase Ⅱ（CA Ⅱ） is an important enzyme involving in various metabolic activities and pathological activity. A gene coding for carbonic anhydrase Ⅱwas cloned from human placenta and an expression plasmid was constructed with plasmid p PIC9 K and introduced into a strain of Pichia. pastoris GS115 by electro-transformation to obtain an engineered strain. The recombinant CA Ⅱ was heterologously overexpressed in the engineered strain GS115 by induction with methanol and purified by anion-exchange chromatography. The purified recombinant CA Ⅱ was verified to have very similar characteristics to a commercial CA Ⅱ through Western blotting,enzyme activity analysis and circular dichroism spectra. This work can lay a foundation for the clinical research and industrial application of CA Ⅱ in the future.

关 键 词：碳酸酐酶Ⅱ 毕赤氏酵母 异源表达 甲醇诱导 

分 类 号：Q78[生物学—分子生物学]