人脑钠肽融合蛋白(HSA-(BNP)_2)在毕赤酵母中的发酵条件及纯化研究  

Fermentative Optimization and Purification of Fusion Protein HSA-(BNP)_2 in Pichia Pastoris

作　　者：丁月娣 刘天会 陈蕴 张莲芬 金坚 

机构地区：[1]江南大学医药学院分子药理研究室,江苏无锡214122

出　　处：《药物生物技术》2009年第2期113-117,共5页Pharmaceutical Biotechnology

基　　金：国家"863"计划项目(2006AA02Z153);上海市科委生物医药重大科技攻关项目(06DZ19020)

摘　　要：对毕赤酵母工程菌GS115/pPIC9K-HSA-(BNP)2的发酵条件进行了优化,并对表达产物HSA-(BNP)2融合蛋白的纯化工艺进行了摸索。研究表明,菌体生长28h时菌浓较高,可进行诱导;同时,在2%甲醇浓度、培养液浓缩1.5倍、诱导表达72h时融合蛋白产量可达185mg/L。发酵液超滤浓缩后经Q Sepharose强阴离子交换层析和Superdex75凝胶过滤层析,获得的融合蛋白纯度为93%,Western blot分析其既具有BNP免疫原性又具有HSA免疫原性。毕赤酵母工程菌GS115/pPIC9K-HSA-(BNP)2经发酵优化后能高效表达HSA-(BNP)2融合蛋白,经两步层析获得的融合蛋白满足后期药效学与药代动力学实验的要求,为后续研究奠定了基础。The fermentation conditions of pichia pastoris GS115/pPIC9K-HSA-（BNP）2 as well as the purification parameters have been optimized. It showed that the strain should be cultured for 28h and then started to induce expression of fusion protein, for the strain is energetic with high density at that moment by the detection of its growth curve. The production of fusion protein could reach 185 mg/L when the strain which has been enriched 1.5 multiplication was induced by 2% methanol for 72 h. The fermentation supernatant was first concentrated by uhrafiltration and then separated by Q Sepharose ion exchange chromatography and Superdex 75 gel filtration chromatography. The interested protein was 93% purity and has both antigenicities of BNP and HSA. The fusion protein of HSA-（BNP）2 could be highly expressed in pichia pastoris GS115/pPICgK-HSA-（BNP）2 after optimization of the fermentation conditions, and it could be used for the study of pharmacodynamics and pharmacokinetics after being purified by two steps of chromatography.

关 键 词：脑钠肽 融合蛋白 毕赤酵母 发酵优化 纯化 

分 类 号：TQ92[轻工技术与工程—发酵工程]