PRODUCTION, PURIFICATION, AND CHARACTERIZATION OF RECOMBINANT 2',5'-OLIGOADENYLATE SYNTHETASES

2',5'-Oligoadenylate [2-5(A)] synthetases are a family of interferon-i nduced enzymes that polymerize ATP into 2'-5'-linked oligoadenylates i n the presence of double-stranded RNA (dsRNA), their cofactor, The 2-5 (A) molecules, in turn, activate the latent ribonuclease RNase L by pr omoting its dimerization. The 2-5(A) synthetase pathway has been impli cated in interferon's antiviral and anticellular activities, In additi on to their interesting cellular properties, these enzymes are also en zymologically interesting because they are the only known template and primer independent nucleotide (DNA or RNA) polymerases that synthesiz e 2'-5'-linked oligonucleotides. Moreover, their mode of activation by dsRNA remains unknown. In the past, biochemical and structure-functio n studies have been hampered by the lack of a convenient system for ex pressing recombinant 2-5(A) synthetases. These proteins are toxic to m ammalian cells, probably because of RNase L activation, and proteins p roduced in bacteria do not have full enzymatic activity. To circumvent these problems, we have developed a baculovirus-insect cell system fo r high-yield expression of the small and medium isozymes. Here, method s are described for the production, purification, and characterization of the mouse small (9-2) (S, K. Ghosh, J. Kusari, S. K. Bandyopadhyay , H. Samanta, R. Kumar, and G. C. Sen, 1991, J. Biol. Chem. 266, 15293 -15299) and human medium (P69) (I. Marie and A. G. Hovanessian, 1992, J. Biol. Chem. 267, 9933-9939) 2-5(A) synthetase isozymes and their mu tants using the insect cell system. We also report methods for studyin g 2-5(A) synthetase-dsRNA interactions and protein-protein interaction s among the subunits of the two isozymes, (C) 1998 Academic Press.