Protein self-splicing and its modulation

Protein splicing is a unique post-translational processing event which involves the removal of an internal polypeptide (the intein) from a precursor protein and the ligation of the two external polypeptides (the exteins). Some inteins have also been shown to be homing endonucleases which arc also found in many introns a.nd are required for intron/intein mobility. We have demonstrated that in t,itTv splicing of inteins from both extreme thermophites and mesophiles is self-catalyzed. Mutagenesis and chemical analysis lead to the identification of key intermediates in the splicing pathway and the definition of the catalytic roles of the four highly conserved splice junction residues (Ser/Cys at the intein N-terminus and His-Asn-(Ser/Cys/Thr) at the C-terminal sptice junction). Deletion and mutagenesis studies suggest that intein elements consist of two functionally and structurally independent endonuclease and splicing domains. Based on an understanding of the chemical mechanism, protein engineering allows us to control splicing and cleavage reactions by amino acid substitutions. A versatile protein purification method based on an intein-catalyzed cleavage reaction has been developed for purification of recombinant proteins via a single chromatographic step.