Single amino acid substitutions in recombinant plant-derived human α1-proteinase inhibitor confer enhanced stability and functional efficacy

Background: Human ai-proteinase inhibitor (alpha 1PI) is the most abundant serine protease inhibitor in the blood and the heterologous expression of recombinant alpha(1)-PI has great potential for possible therapeutic applications. However, stability and functional efficacy of the recombinant protein expressed in alternate hosts are of major concern. Methods: Five variants of plant-expressed recombinant alpha(1)-PI protein were developed by incorporating single amino acid substitutions at specific sites, namely F51C, F51L, A70G, M358V and M374I. Purified recombinant alpha(1)-PI variants were analyzed for their expression, biological activity, oxidation-resistance, conformational and thermal stability by DAC-ELISA, porcine pancreatic elastase (PPE) inhibition assays, transverse urea gradient (TUG) gel electrophoresis, fluorescence spectroscopy and far-UV CD spectroscopy. Results: Urea-induced unfolding of recombinant alpha(1)-PI variants revealed that the F51C mutation shifted the mid-point of transition from 1.4 M to 4.3 M, thus increasing the conformational stability close to the human plasma form, followed by F51L, A7OG and M374I variants. The variants also exhibited enhanced stability for heat denaturation, and the size-reducing substitution at Phe51 slowed down the deactivation rate similar to 5-fold at 54 degrees C. The M358V mutation at the active site of the protein did not significantly affect the conformational or thermal stability of the recombinant alpha(1)-PI but provided enhanced resistance to oxidative inactivation. Conclusions: Our results suggest that single amino acid substitutions resulted in improved stability and oxidation-resistance of the plant-derived recombinant alpha(1)-PI protein, without inflicting the inhibitory activity of the protein. General significance: Our results demonstrate the significance of engineered modifications in plant-derived recombinant alpha(1)-PI protein molecule for further therapeutic development. (C) 2013 Elsevier B.V. All rights reserved.