The consensus-based approach for gene/enzyme replacement therapies and crystallization strategies: the case of human alanine-glyoxylate aminotransferase.

[primary hyperoxaluria type 1]

Protein stability is a fundamental issue in biomedical and biotechnological applications of proteins . Among these applications , gene - and enzyme-replacement strategies are promising approaches to treat inherited diseases that may benefit from protein engineering techniques , even though these beneficial effects have been largely unexplored . In the present study we apply a sequence-alignment statistics procedure (consensus-based approach ) to improve the activity and stability of the human AGT (alanine-glyoxylate aminotransferase ) protein , an enzyme which causes PH 1 (primary hyperoxaluria type I ) upon mutation . By combining only five consensus mutations , we obtain a variant (AGT -RHEAM ) with largely enhanced in vitro thermal and kinetic stability , increased activity , and with no side effects on foldability and peroxisomal targeting in mammalian cells . The structure of AGT -RHEAM reveals changes at the dimer interface and improved electrostatic interactions responsible for increased kinetic stability . Consensus-based variants maintained the overall protein fold , crystallized more easily and improved the expression as soluble proteins in two different systems [AGT and CIPK 24 (CBL -interacting serine /threonine-protein kinase ) SOS 2 (salt -overly-sensitive 2 )]. Thus the consensus-based approach also emerges as a simple and generic strategy to increase the crystallization success for hard-to -get protein targets as well as to enhance protein stability and function for biomedical applications .