An intein from Halobacterium salinarum can be isolated as an unspliced precursor protein with exogenous exteins after Escherichia coli overexpression. The intein promotes protein splicing and uncoupled N-terminal cleavage in vitro, conditional on incubation with NaCl or KCl at concentrations of >1.5 M. The protein splicing reaction also is conditional on reduction of a disulfide bond between two active site cysteines. Conditional protein splicing under these relatively mild conditions may lead to advances in intein-based biotechnology applications and hints at the possibility that this H. salinarum intein could serve as a switch to control extein activity under physiologically relevant conditions.

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Protein splicing is the self‐catalyzed excision of an intervening polypeptide (intein) from flanking polypeptides (exteins), concomitant with the ligation of the extein. An intein in the archaebacterium Halobacterium salinarum (Hsa) interrupts the DNA Polymerase II. We have observed that the Hsa intein splices poorly in Escherichia coli cells, unlike two highly related inteins found in Pyrococcus abyssi and Methanoculleus marisnigri, which are not halophilic. To test if splicing activity depends on salinity, we altered the expression conditions by varying the salinity of the growth media and of in vitro activity assays. Future experiments will involve in vivo techniques by over‐expressing the intein and the flanking exteins into Hsa, and by using antibodies to detect possible splicing of the native protein. Grant Funding Source: Supported by the National Science Foundation under grant MCB‐1244089 and by the Dreyfus Foundation

Protein splicing is a self‐catalyzed process where an intervening intein is excised, with the concomitant ligation of the two flanking exteins to form a mature protein. The inteins we studied interrupt the DNA Pol II of Pyrococcus abyssi and Pyrococcus horikoshii. These extremophile inteins conditionally splice at high temperatures. The Hyperthermophilic and Thermophilic Hairpin (HTH) is only found in the structures of archaeal inteins. We hypothesize that the HTH increases stability by elongating the central beta‐sheet, and thus mutations that disrupt this hairpin would affect the stability toward unfolding and the reactivity at the active site. To further understand the protein splicing of the P. abyssi intein, an intein with an Arg40Glu mutation in the HTH was expressed, and we studied its activity. We also purified 15‐N labeled intein for NMR (HSQC) analysis of residue‐level changes induced by the mutation. We have also purified a variant of the P. horikoshii PolII intein in order to attempt to solve the structure of an unspliced precursor intein.Support or Funding InformationThis work was supported by the National Science Foundation (grant MCB‐1517138) and by the Camille & Henry Dreyfus FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Protein splicing is the self‐catalyzed excision of an intervening polypeptide (intein) from flanking polypeptides (exteins), concomitant with the ligation of the exteins. Similar inteins found in two thermophilic archaebacteria, Pyrococcus abyssi (Pab) and Pyrococcus horikoshii (Pho), interrupt the DNA Polymerase II. Comparison of the Pab PolII NMR structure and a Pho PolII homology model, along with 75% sequence identity between the inteins, reveals a common fold with the exception of a longer, disorganized loop in the Pab intein. The longer loop may provide a less rigid structure that allows splicing to occur at lower temperatures. Both splicing and uncoupled cleavage at the intein junctions occur to a greater extent at lower temperatures for the Pab intein than with the Pho intein. The relative rigidity of the inteins was compared by treatment of each with the thermostable protease thermolysin, which cleaves proteins in flexible regions. Thermolysin cleavage was more efficient at lower temperatures for the Pab intein. We are currently examining the relative strength of a disulfide bond that links the catalytic Cys residues as a measure of the flexibility of both inteins' active sites by titration with DTT.