Abstract 1265 Mutants on Mars: Directed Evolution of Halophile Enzyme Activity

Protein splicing is the process by which intervening segments of protein called inteins can self-excise themselves out of the protein and catalyze the ligation of the flanking exteins. In Halobacterium salinarum (Hsa), a halophilic archaeon, this splicing occurs only at extremely high salt concentrations. We are interested in determining what residues are responsible for the salt dependence of both the folding and the activity of the H. salinarum intein that interrupts the DNA polymerase II. We are undertaking a directed evolution strategy, coupling protein splicing to kanamycin resistance by interrupting the kanamycin resistance protein with an intein. E. coli transformed with a plasmid that expresses this interrupted kanamycin resistance gene grows best in the presence of no kanamycin, moderately well at 5 and 10 μg/ml of kanamycin, and grows poorly or does not grow at concentrations above 15 μg/ml. Mutations in this gene can be made through error-prone PCR and the resulting DNA will be transformed into E. coli cells to be plated on different levels of kanamycin. This allows us to select mutants that are able to splice at high antibiotic concentrations and therefore likely can promote intein splicing at lower salt concentrations. We will progressively create a version of a halophile intein that requires less salt to determine which residues of the intein are important for salt dependence. We thank the National Institutes of Health, NIGMS (Grant 1R15GM132817-01) for supporting our research.