Alternate protein splicing mechanisms: A directed evolution approach

Abstract

Inteins are intervening polypeptides that are able to direct their excision from the flanking polypeptides, called exteins, as well as facilitate the ligation of the exteins. This process, called protein splicing, produces the functional protein. We want to determine if inteins that splice by the standard mechanism also can splice via alternate mechanisms. First, we have inactivated splicing of the RecA intein from M. tuberculosis by mutating the N‐terminal Cys to Ala. Using saturation mutagenesis and error prone PCR, we generated random mutants that we subjected to a genetic screen to select for complementary mutations that may allow the modified intein to facilitate splicing by bypassing the first step. To select for complementary mutants, we used E. coli harboring a plasmid with the kanamycin resistance gene, aminoglycoside phosphotransferase, interrupted by the RecA intein, such that bacterial growth should depend on efficient splicing. We have been able to mutate the RecA intein and select inteins that allow for growth on kanamycin. However, these inteins do not promote splicing in our biochemical assay, which suggests that the intein may allow for a functional aminoglycoside phosphotransferase in the absence of splicing. This material is based upon work supported by the National Science Foundation under Grant No. 0320824 and CAREER grant No. 0447647.