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

Abstract:

Abstract: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-pPICZα  expression 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-pPICZα expression 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 6.0 and 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.

Key words: Pichia , pastoris；rhKD/APPvar；bovine pancreatic trypsin inhibitor；amyloid protein precursor