A Directed Evolution Experiment to Improve Protein Splicing of Two Inteins From Haloquadratum walsbyi.

We are interested in the protein splicing of the inteins from the extreme halophile Haloquadratum walsbyi. Protein splicing is a post translational process by which an intein excises itself and ligates the two flanking polypeptides, the exteins. Two inteins interrupt the cell division control (cdc21) protein in H. walsbyi. One intein splices only at salt concentrations above 2 M, whereas the other will splice at lower salt concentrations. We have designed a directed evolution experiment coupling the growth of E. coli to successful splicing of each intein from the protein that confers resistance to the antibiotic kanamycin. Preliminary results suggest that each intein can promote splicing of the kanamycin resistance protein, and we are currently testing a pool of intein mutants for enhanced splicing activity.