Production of Two Human 2′,5′-Oligoadenylate Synthetase Enzymes in Escherichia coli

Abstract

We have isolated and characterized two types of cDNA clones corresponding to interferon (IFN)-induced 1.6- and 1.8-kb mRNAs, as encoding two different forms of the 2′,5′-oligoadenylate (2′-5′)A synthetase enzyme. Direct expression of the two cDNAs was obtained in Escherichia coli under the control of a trp-lac hybrid promoter strongly inducible in E. coli by IPTG. Bacterial extracts were tested for 2′-5′A synthetase activity after adsorption to immobilized poly (I)•poly(C) or in solution. With either one of the cDNA constructions, IPTG induced 2′-5′A synthetase activity in the bacteria to levels 10 times higher per microgram of protein than those in SV80 cells treated by 500 U/ml of IFN-β₁ for 24 h. Both bacterially produced enzymes bind to double-stranded (ds)RNA and are maximally active at 100 μg/ml of poly(I)•poly(C). Both enzymes synthesized similar 2′-5′(Ap) _n A oligomers of 2 to 8 residues in length. Antibodies against a synthetic peptide common to the two enzymes were used to characterize the bacterial products on immunoblots and confirmed that the 1.6-kb RNA produces a 39-kD protein, whereas the 1.8-kb RNA encodes a 45- to 46-kD protein. The E. coli enzyme coded by the 1.6-kb mRNA was purified to nearly homogeneity. When immobilized on poly(I)•poly(C) agarose, the enzyme produces, per milliliter of poly(I)•poly(C), 10³ times more 2′-5′(Ap) _n A oligomer than the most active cellular extracts. Moreover, the immobilized enzyme remains stable for several months at 4°C.