Enzymatic characteristics of recombinant medium isozyme of 2 '-5 ' oligoadenylate synthetase

P69 is an isozyme of the medium size class of human 2'-5' oligoadenylate sy nthetases. In this study, recombinant P69 was expressed and used for enzymo logical and structural investigations. Bacterially expressed P69 was inacti ve whereas the same protein expressed in insect cells was highly active. Wh ether this difference could be due to differential post-translational modif ications of the protein was investigated. Mutations of appropriate residues showed that myristoylation of the protein was not necessary for enzyme act ivity. In contrast, inhibition of glycosylation of P69, by tunicamycin trea tment of the insect cells, produced an enzymatically inactive protein, Reco mbinant P69 produced in insect cells was purified by affinity chromatograph y. It was a dimeric glycoprotein, very stable and completely dependent on d ouble stranded (ds) RNA for activity. The enzyme catalyzed the non-processi ve synthesis of 2'5'-linked oligoadenylate products containing up to 30 res idues, 2'-O-Methylated dsRNA was incapable of activating P69 and a 25-base pair dsRNA was as effective as larger dsRNA. This expression system will be useful for large scale production of P69 and its mutants for structural st udies.