Purification And Purity Identification Of Recombinant Protein Rh(GLP-1_A2G)₂-HSA

Intensive studies on the process of fermentation, purification and bioactivity were conducted in this thesis.During the fermentation, the producing stain Pichia Pastoris KM71/(GLP-1_A2G)₂-HSA was induced after 60 hours processing, then the concentration of (GLP-1_A2G)₂-HSA in the fermentation liquid reached 210 mg/L. The optimized expression condition consisted temperature 30℃, pH 6.0 and the methanol induced concentration 2.0 %.This research mainly dealt with purification of recombinant protein rh(GLP-1_A2G)₂-HSA from fermentation broth. Highly purified rh(GLP-1_A2G)₂-HSA was separated from fermentation broth by ultrafiltration concentration, ion exchange chromatography and gel filtration. The purified product was conformed as one single band by SDS-PAGE and its purity was identified as 98% by HPLC while its radioactive purity was identified as 97% by TCA deposition method after marked with 125I. The purity of rh(GLP-1_A2G)₂-HSA met the requirement of drug activity and drug metabolism research. The total recovery yield reached 48.5 %. Highly purified rh(GLP-1_A2G)₂-HSA could be gained by this purification method and layed a foundation for advanced drug activity and drug metabolism research.Cell proliferative assay showed that the activity of rh(GLP-1_A2G)₂-HSA remained well during the purification while the purified product had similar proliferative activity of isletβ-cell with GLP-1. When the concentration of rh(GLP-1_A2G)₂-HSA is 40 nmol/L, the growth rate is 35.4%.