Expression and purification of functionally active recombinant human alpha 1-antitrypsin in methylotrophic yeast pichia pastoris

ABSTRACT

Human alpha 1-antitrypsin [AAT] cDNA was obtained from HepG2 cell lines. After PCR and construction of expression vector pPICZ?-AAT, human AAT was expressed in the yeast Pichia pastoris [P.pastoris] in a secretary manner and under the control of inducible alcohol oxidase 1 [AOX1] promoter. The amount of AAT protein in medium was measured as 60 mg/l 72 hr after induction with methanol. Results indicated the presence of protease inhibitory function of the protein against elastase. Purification was done using His-tag affinity chromatography. Due to the different patterns of glycosylation in yeast and human, the recombinant AAT showed different SDS-PAGE patterns compared to that of serum-derived AAT while pI shifted from 4.9 in native AAT compared to 5.2 in recombinant AAT constructed in this study