摘要
目的：在毕赤酵母（Pichia pastoris）中高效表达AβPP中的Kunitz型蛋白酶抑制剂的结构域（KPI／AβPP），利用80L的发酵罐优化发酵条件，制备rhKPI／AβSPP。方法：克隆kpi基因插入到pPICZα，构建真核表达载体pPICZμ-kpi，经核酸测序确证后，电转化毕赤酵母菌株X-33，筛选高表达rhKPI／AβPP工程菌。结果：筛选出的高表达工程菌采用80L发酵罐甲醇诱导补料批式发酵，在pH3．3、纯氧浓度22％～30％、罐内压力为12psi、甲醇诱导60h时产量1．0g·L^-1。纯化的rhKPI／AβPP经SDS-PAGE分析显示单一区带，相对分子质量6750。质谱测定其相对分子质量为6750，抑制胰蛋白酶的比活性为8．4EPU·mg^-1。结论：克隆、构建、筛选出高效表达rhKPI／AβPP的毕赤酵母工程菌，并建立了稳定的发酵和纯化工艺。
Objective To study the efficient expression of a Kunitz-type protease inhibitor （KPI） domain of the Alzheimer＇s disease related to amyloid β-protein precursor, and produce rhKPI/AβPP by optimizing fermentation papameters on large scale using Pichia pastoris as host system. Methods KPI/AβPP gene was inserted into expression vector pPICZa and the resulted recombinant vector was transformed into the Pichia pastoris. Then KPI/AβPP was expressed in 80L fermentor with optimizing parameters and the broth was harvested and purified with SP Sepharose. Results KPI/AβPP gene could be induced by methanol and expressed with the maximal yield of 1.0 g · L^-1. The expressed product was purified from the fermentating culture. Analytic results revealed that resulted product with relative molecular mass of about 6 750 inhibited the serine protease activities. Conclusion rhKPI/AβPP can be expressed in Pichia pastoris on large scale. A stable fermentation and purification technology is successfully set up.
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2006年第2期278-281,共4页
Journal of Jilin University：Medicine Edition
基金
国家高技术研究发展计划(863计划)资助课题(2004AA205020)
