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

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 120 h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。

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

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 Pichia pastoris,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 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 6.0and the optimal induction time point was about the 5th 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.