Construction of secretory expression vector of rhKD/APPvar and expression and purification of its recombinant protein in Pichia pastoris
10.13481/j.1671-587x.20140313
- VernacularTitle: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
- Author:
Xintong WANG
;
Hongjiao WANG
;
Qiang WANG
;
Weihong MENG
;
Weiqun YAN
;
Liqun REN
- Publication Type:Journal Article
- Keywords:
Pichia pastoris;
rhKD/APPvar;
bovine pancreatic trypsin inhibitor;
amyloid protein precursor
- From:
Journal of Jilin University(Medicine Edition)
2014;(3):529-533
- CountryChina
- Language:Chinese
-
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.
