PRODUCTION AND PURIFICATION OF RECOMBINANT 2'-5'-OLIGOADENYLATE SYNTHETASE AND ITS MUTANTS USING THE BACULOVIRUS SYSTEM

Investigation of the structure-function relationship of the 2'-5' olig oadenylate [2-5 (A)] synthetases has been hampered by the lack of an e fficient expression system for a recombinant enzyme. Here, we report t hat the 9-2 isozyme of murine 2-5 (A) synthetase can be efficiently ex pressed in insect cells using the baculovirus system. The recombinant protein was purified to apparent homogeneity, and its enzymatic activi ty was characterized. It had a high specific activity, required double -stranded RNA as a cofactor, and synthesized dimers to hexamers of 2-5 (A). The utility of our expression system was demonstrated by studyin g the properties of two previously reported mutant proteins. Both of t hese mutants, when produced in bacteria, are enzymatically inactive, a lthough similarly produced wild-type protein is active. Unexpectedly, when expressed in insect cells, both mutant proteins were enzymaticall y as active as the wild-type protein. These results suggest that in th e eukaryotic expression system described here, the mutant proteins can undergo appropriate modifications or folding that is required for att aining an enzymatically active conformation.