Towards Bacterial Expression of Unstable Mutants of a Mitochondrial Enzyme

Abstract

Mutations in the human AARS2 gene, which codes for the mitochondrial alanyl‐tRNA synthetase, are linked with severe disease, including infantile cardiomyopathy and leukodystrophy. Our laboratory is interested in understanding, on a molecular level, why different mutations result in different disease phenotypes. Attempts to produce some mutants of AARS2 using a bacterial expression system have resulted in poor expression of proteins contaminated with proteolytic fragments. To improve expression of these mutants, we have used solubility tags to facilitate the purification of full‐length mutant protein. Addition of an MBP solubility tag greatly improved expression and purification of the wildtype and mutant enzymes. We are currently investigating strategies for solubility tag removal and characterization of wildtype and mutant thermal stability.