Intein‐mediated peptide bond cleavage adjacent to aspargine or glutamine

Abstract

Protein splicing is a post‐translational, self‐catalyzed process in which an intervening polypeptide, the intein, is self‐excised from the flanking polypeptides, or exteins. The intein that interrupts the DNA polymerase of Pyrococcus abyssi can facilitate protein splicing in a temperature‐dependent fashion. This intein has a Cterminal Gln, which facilitates the third step of splicing, side chain cyclization coupled to peptide bond cleavage. We plan to induce C‐terminal cleavage of an intein (uncoupled from splicing) to facilitate purification from a solid affinity resin. We have fused the intein with an N‐terminal affinity domain and a C‐terminal target protein for purification, such that C‐terminal cleavage of the intein via cyclization of Asn or Gln liberates the C‐terminal domain with an N‐terminal Cys. This fragment will serve as a substrate for expressed protein ligation. In addition, we will compare the rate of the cyclization between glutamine and asparagine in model peptides with an N‐terminal Abz fluorophore and a C‐terminal Lys‐DNP quencher, using a fluorescence assay to measure cleavage. We will determine the rate of cleavage as a function of temperature and pH.This material is based upon work supported by the National Science Foundation under grant MCB‐0950245 and by the Camille and Henry Dreyfus Foundation.