Purification of recombinant proteins from E. coli by engineered inteins.

Abstract

The IMPACT (Intein-Mediated Purification with an Affinity Chitin-Binding Tag) vectors are designed for the isolation of pure, functional, recombinant proteins by a single affinity chromatography step. The IMPACT technology was developed at New England Biolabs (NEB) by exploiting a novel family of proteins termed inteins (recently reviewed in ref. ). An intein is an internal protein segment responsible for catalyzing an extraordinary post-translational processing event termed protein splicing. Protein splicing results in the precise excision of the intein polypeptide from a protein precursor with the concomitant ligation of the flanking protein sequences, termed exteins. This process requires neither auxiliary proteins nor exogenous energy sources such as ATP (for more information on the requirements and mechanism of protein splicing, see ref. ). Once the mechanism of protein splicing was elucidated it was realized that a self-splicing intein could be used for protein purification, because the catalytic steps involved in the fission of the peptide bond at either splice junction could be modulated by mutation of amino acid residues at the splice junctions, as described in detail in the following sections.