Production of two human 2',5'-oligoadenylate synthetase enzymes in Escherichia coli.
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-beta1 for 24 h. Both bacterially produced enzymes bind to double-stranded (ds)RNA and are maximally active at 100 micrograms/ml of poly(I).poly(C). Both enzymes synthesized similar 2'-5'(Ap)nA 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), 10(3) times more 2'-5'(Ap)nA oligomer than the most active cellular extracts. Moreover, the immobilized enzyme remains stable for several months at 4 degrees C.
Mory
Y
Y
Department of Virology, Weizmann Institute of Science, Rehovot, Israel.
Vaks
B
B
Chebath
J
J
D015088:2',5'-Oligoadenylate Synthetase / Q000096:biosynthesis* / Q000235:genetics; D002852:Chromatography, Ion Exchange; D002999:Clone Cells; D003001:Cloning, Molecular; D004270:DNA, Circular / Q000302:isolation & purification*; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015151:Immunoblotting; D010957:Plasmids
D004270:DNA, Circular; D004274:DNA, Recombinant; D015088:2',5'-Oligoadenylate Synthetase