Rapid purification of a cloned gene product by genetic fusion and site-specific proteolysis.

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Abstract
A rapid and general technique was developed for purification of a protein encoded by a cistron contained in a recombinant DNA clone. The technique consists of fusing the target cistron DNA in the correct reading frame to a marker cistron via a piece of DNA that codes for a linker peptide. The target cistron in the example presented here is the replication initiator cistron of the plasmid R6K. The linker is a DNA fragment encoding 60 amino acids from the triple helical region of chicken pro.alpha.-2 collagen, and the marker cistron encodes the .beta.-galactosidase protein of Escherichia coli. The tripartite hybrid protein was rapidly purified by selective binding to and elution from a .beta.-galactosidase specific-affinity column. The hybrid protein was then digested with a purified microbial collagenase to cleave the linker, and high-pressure liquid chromatography allowed the rapid isolation of the target protein from the marker protein. Using this technique, the highly labile R6K replication initiator was purified to homogeneity. The protein was resolved into NH2-terminal and COOH-terminal segments. By in vitro binding, the COOH-terminal segment was found to have at least 1 DNA-binding domain. The domain binds to the same restriction fragments of the R6K chromosome as the intact or .beta.-galactosidase-tagged initiator protein.