Homing Endonuclease and Protein Splicing Activity of Inteins from Thermophilic Archaea

Abstract

Protein splicing is a post translational, self‐catalyzed reaction by which an intein removes itself from flanking polypeptides and ligates those polypeptides together. We are interested in the splicing and homing endonuclease activities of the intein that interrupt the DNA polymerase II in the extreme thermophiles Thermococcus barophilus and Thermococcus kodakarensis. Given that T. kodakarensis is a surface‐dwelling organism whereas the T. barophilus is a piezophile that lives in deep‐sea thermal vents, yet both inteins are highly similar in sequence, we are interested in how temperature and pressure may affect enzyme activity. Additionally, we are interested in the role of the two conserved C‐terminal residues of the intein. We have found that the homing endonuclease domain of the T. kodakarensis intein is more active at a lower concentration and lower temperatures than that of the T. barophilus intein. Protein splicing of both inteins can be induced by in vitro incubation of an isolated, unspliced precursor at elevated temperature, which may also be influenced by hydrostatic pressure. Support or Funding Information This work was supported by NSF grant MCB‐1517138, a Henry Dreyfus Teacher‐Scholar Award, and by NIH Grant 1R15GM132817‐0