SINGLE-COLUMN PURIFICATION OF FREE RECOMBINANT PROTEINS USING A SELF-CLEAVABLE AFFINITY TAG DERIVED FROM A PROTEIN SPLICING ELEMENT

A novel protein purification system has been developed which enables p urification of free recombinant proteins in a single chromatographic s tep. The system utilizes a modified protein splicing element (intein) from Saccharomyces celevisiae (Sce VMA intein) in conjunction with a c hitin-binding domain (CBD) from Bacillus circulans as an affinity tag. The concept is based on the observation that the modified See VMA int ein can be induced to undergo a self-cleavage reaction at its N-termin al peptide linkage by 1,4-dithiothreitol (DTT), beta-mercaptoethanol ( beta-ME) or cysteine at low temperatures and over a broad pH range. A target protein is cloned in-frame with the N-terminus of the intein-CB D fusion, and the stable fusion protein is purified by adsorption onto a chitin column. The immobilized fusion protein is then induced to un dergo self-cleavage under mild conditions, resulting in the release of the target protein while the intein-CBD fusion remains bound to the c olumn. No exogenous proteolytic cleavage is needed. Furthermore, using this procedure, the purified free target protein can be specifically labeled at its C-terminus. (C) 1997 Elsevier Science B.V.