EFFICIENT AND RAPID AFFINITY PURIFICATION OF PROTEINS USING RECOMBINANT FUSION PROTEASES

In the affinity purification of recombinant fusion proteins, the rate- limiting step is usually the efficient proteolytic cleavage and remova l of the affinity tail and the protease from the purified recombinant protein. We have developed a rapid, convenient and efficient method of affinity purification which can overcome this limitation. In one exam ple of the method, the protease 3C from a picornavirus (3C(pro)), whic h cleaves specific sequences containing a minimum of 6-7 amino acids, has been expressed as a fusion with glutathione S-transferase. The res ultant recombinant 'fusion protease' cleaves fusion proteins bearing ( from the amino-terminus) the same affinity tail as the fusion protease , a 3C(pro) cleavage recognition site, and the recombinant protein of interest. The recombinant protein is purified in a single chromatograp hic step which removes both the affinity tail and the fusion protease. The advantages over existing methods include much improved specificit y of proteolytic cleavage, complete removal of the protease and the af finity tail in one step, and the option of adding any desired amount o f fusion protease to ensure efficient cleavage. The potential flexibil ity of the method is shown by the use of various affinity tails and al ternative fusion proteases.