The crystal structure of the naturally split gp41-1 intein guides the engineering of orthogonal split inteins from a cis-splicing intein

Protein trans-splicing catalyzed by split inteins has increasingly become useful as a protein engineering tool. The 1.0 A-resolution crystal structure of a variant from naturally split gp41-1 intein, identified from the environmental metagenomic sequence data, revealed an improved pseudo-C2-symmetry commonly found in the Hedgehog/Intein (HINT) superfamily with extensive charge-charge interactions between the split N- and C-terminal intein fragments. We successfully created orthogonal split inteins by engineering a similar charge network in the same region of a cis-splicing intein. The same strategy could be applicable for creating novel natural-like split inteins from other, more prevalent cis-splicing inteins.