An intein is an intervening protein segment that can be found in proteins from extremophiles and is typically self‐excised to allow for complete flanking protein activity. We are interested in ...studying specific inteins that contain a homing endonuclease (HE), an enzyme that cleaves double stranded DNA in order to initiate insertion of an intein gene by homologous recombination. We are focused on the homing endonuclease activity of inteins from two hyperthermophilic archaea: Thermococcus barophilus (Tba), isolated from a deep sea hydrothermal vent, and Thermococcus kodakarensis (Tko), isolated from a volcanic area on the shore of a Japanese island. Because these intein‐containing extremophiles have been isolated from very high temperatures but from different depths, we are interested in the differential homing endonuclease activity of these extremophiles at varied temperatures and pressures. We have shown that Tba HE is active at pressures up to 1000 atm. Given that Tko is a surfaced‐welling organism, we suspect that the Tko HE will be pressure sensitive while Tba, which lives naturally at a pressure near 400 atm, is not. Additionally, we have found that both Tba HE and Tko HE show temperature dependence, which we attribute to the extreme temperatures that both organisms reside in.
Support or Funding Information
This work was supported by the National Science Foundation (grant MCB‐1517138) and by the Camille & Henry Dreyfus Foundation.
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.
Abstract only
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
PDF
