PROTEIN SPLICING IN TRANS BY PURIFIED N-TERMINAL AND C-TERMINAL FRAGMENTS OF THE MYCOBACTERIUM-TUBERCULOSIS RECA INTEIN

Protein splicing involves the self catalyzed excision of protein splic ing elements, or inteins, from flanking polypeptide sequences, or exte ins, leading to the formation of new proteins in which the exteins are linked directly by a peptide bond, To study the enzymology of this in teresting process we have expressed and purified N- and C-terminal seg ments of the Mycobacterium tuberculosis RecA intein, each approximate to 100 amino acids long, fused to appropriate exteins, These fragments were reconstituted into a functional protein splicing element by rena turation from 6 M urea, When renaturation was carried out in the absen ce of thiols, the reconstituted splicing element accumulated as an ina ctive disulfide-linked complex of the two intein fragments, which coul d be induced to undergo protein splicing by reduction of the disulfide bond, This provided a useful tool for separately investigating the re quirements for the reconstitution of the intein fragments to yield a f unctional protein splicing element and for the protein splicing proces s per se, For example, the pH dependence of these processes was quite different, with reconstitution being most efficient at pH 8.5 and spli cing most rapid at pH 7.0, The availability of such an in vitro protei n splicing system opens the way for the exploration of intein structur e and the unusual enzymology of protein splicing, In addition, this tr ans-splicing system is a potential protein ligase that can link any tw o polypeptides fused to the N and C-terminal intein segments.