Protein splicing is a self‐catalyzed reaction by which an intein, an intervening polypeptide, catalyzes its own removal from exteins, the flanking polypeptides, as well as ligation of the exteins. We are studying the inteins that interrupt a cell division control protein in the halophilic archaea Haloquadratum walsbyi. Halophiles like H. walsbyi are able to survive in water with high concentrations of sodium chloride. H. walsbyi has four inteins that interrupt the cdc21 gene, and we have studied the two inteins (Hwa cdc21A and Hwa cdc21D) that lack an intervening homing endonuclease domain. We are studying the splicing activity under various salt concentrations for the inteins both separately and when expressed as part of a single fusion protein. Hwa cdc21A splices optimally at 2.5 M NaCl, while Hwa cdc21D splices optimally at 1.5 M NaCl. We also are using native Trp fluorescence to study the influence of salt concentration on the proper folding of the inteins. Support or Funding Information This work was supported by NSF grant MCB‐1517138, a Henry Dreyfus Teacher‐Scholar Award, and NIH Grant 1R15GM132817‐01.

Protein splicing is a post-translational process by which an intervening protein, or an intein, catalyzes its own excision from flanking polypeptides, or exteins, coupled to extein ligation. Four inteins interrupt the MCM helicase of the halophile Haloquadratum walsbyi, two of which are mini-inteins that lack a homing endonuclease. Both inteins can be overexpressed in Escherichia coli and purified as unspliced precursors; splicing can be induced in vitro by incubation with salt. However, one intein can splice in 0.5 M NaCl in vitro, whereas the other splices efficiently only in buffer containing over 2 M NaCl; the organism also requires high salt to grow, with the standard growth media containing over 3 M NaCl and about 0.75 M magnesium salts. Consistent with this difference in salt-dependent activity, an intein-containing precursor protein with both inteins promotes conditional alternative protein splicing (CAPS) to yield different spliced products dependent on the salt concentration. Native Trp fluorescence of the inteins suggests that the difference in activity may be due to partial unfolding of the inteins at lower salt concentrations. This differential salt sensitivity of intein activity may provide a useful mechanism for halophiles to respond to environmental changes.

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.