Abstract
Alpha-1 antitrypsin (AAT) is the most abundant member of the serpin superfamily of protease inhibitors found in human plasma. Expression of a broad-spectrum AAT variant (AAT M358R) and an AAT variant (AAT-RC-2) that specifically inhibits coagulation Factor XIa (FXIa) in the methylotrophic yeast Pichia pastoris were compared. When protein secretion was directed by the 85 amino acid Saccharomyces cerevisiae alpha mating factor (AMF) prepro signal sequence 1 (ss1), AAT-RC-2 was purified as a 45 kDa homogeneous polypeptide preparation, but AAT M358R was heterogeneous. Replacement of the ss1 prepro sequence with any of 5 presequences lacking pro sequences eliminated the heterogeneity. The highest yield was obtained with ss3-AAT M358R, where ss3 was the 19 amino acid Saccharomyces cerevisiae AMF presequence. Enzymatic deglycosylation of ss1-AAT M358R converted its high molecular weight heterogeneity into a simple combination of 55 kDa and 45 kDa polypeptides consistent with failure to remove the AMF presequence and inhibition of the Kex2 propeptide convertase by AAT M358R. Purified ss3-AAT M358R inhibited FXIa significantly more rapidly than E. coli-derived AAT M358R with indistinguishable reaction stoichiometry; purified ss1-AAT-RC-2 did not differ from its E. coli-derived counterpart in either kinetic parameter. Both AAT variants formed denaturation-resistant complexes with FXIa. Substitution of the AMF prepro sequence with its constituent presequence eliminated the protein expression problem caused by inhibition of the Pichia pastoris propeptide processing machinery by AAT M358R and will make feasible comparison of AAT M358R and AAT-RC-2 in animal models of thrombosis and bleeding.