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-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 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.