rhKD/APPvar毕赤酵母分泌表达质粒的构建及其重组蛋白的表达和纯化  

Construction of secretory expression vector of rhKD/APPvar and expression and purification of its recombinant protein in Pichia pastoris

作　　者：王心童[1] 王虹蛟 王强 孟威宏[3] 颜炜群[4] 任立群[4] 

机构地区：[1]吉林大学中日联谊医院神经内科,吉林长春130033 [2]解放军第461医院内科,吉林长春130021 [3]沈阳军区总医院心血管内科,辽宁沈阳110015 [4]吉林大学再生医学科学研究所再生医学系,吉林长春130021

出　　处：《吉林大学学报（医学版）》2014年第3期529-533,共5页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金资助课题(30300153)

摘　　要：目的:构建高效分泌表达重组人淀粉样蛋白前体Kunitz型蛋白酶抑制剂结构域变异体(rhKD/APPvar)的毕氏酵母工程菌,建立适合大规模发酵、纯化rhKD/APPvar的工艺。方法:利用已构建的rhKD/APP表达质粒,在其活性中心两侧设计2个酶切位点(ApaⅠ和SacⅡ),实现人KD/APP活性中心RAM与BPTI活性中心KAR的替换,构建rhKD/APPvar表达质粒。将重组质粒转化到酵母菌X-33中,优化rhKD/APPvar表达最佳pH值,使rhKD/APPvar获得高效表达。利用阳离子交换树脂和超滤除盐对重组蛋白进行纯化。结果:酶切鉴定和测序分析显示成功构建了KD/APPvar-pPICZαC重组质粒。经电转化成功地将重组质粒转化至酵母菌X-33中。SDS-PAGE分析,甲醇诱导表达后在相对分子质量约6 700处出现蛋白条带,pH 6.0、甲醇诱导120h蛋白表达水平最高,经纯化获得纯度达95%的重组蛋白。结论:成功构建KD/APPvar-pPICZαC重组质粒,经毕赤酵母表达和纯化获得了rhKD/APPvar蛋白。Objective To construct the engineering bacteria expressing the recombinant human Kunitz protease inhibitor domain of amyloid protein precursor variant （rhKD/APPvar）in Pichia pastoris,and to establish the methods suitable for large-scale fermentation and purification of rhKD/APPvar.Methods The rhKD/APPvar expression vector was constructed based on the rhKD/APPvar-pPICZαexpression vector. Two restriction enzyme loci （ApaⅠ and SacⅡ）were added to two flanks of KD/APP and human KD/APP activity center RAM was replaced by the active site of BPTI KAR.After the rhKD/APPvar-pPICZαexpression vector was transformed into Pichiapastoris,optimized expression and purification of rhKD/APPvar was performed.The rhKD/APPvar was purified with cation exchange chromatography and desalting.Results The results of digestion identification and DNA sequencing analysis demonstrated that the recombinant plasmid rhKD/APPvar-pPICZα was successfully＆amp;nbsp;constructed and transfected into pastoris X-33. The SDS-PAGE analysis results indicated that rhKD/APPvar expressed after the induction of methanol and the relative molecular weight was 6 700.After a series of experiments the optimal expression conditions of rhKD/APPvar were obtained as follows：the optimal pH was 6.0 and the optimal induction time point was about the 5 th day for the strain.After purified the purity of rhKD/APPvar was about 95%.Conclusion KD/APPvar-pPICZ is successfully constructed;after expression in Pichia pastoris and purification,the rhKD/APPvar protein is obtained.

关 键 词：毕赤酵母菌 重组KD APP变异体 牛胰蛋白酶抑制剂 淀粉样蛋白前体 

分 类 号：Q78[生物学—分子生物学]