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' oligoadenylate [2-5 (A)] synthetases has been hampered by the lack of an efficient expression system for a recombinant enzyme. Here, we report that the 9-2 isozyme of murine 2-5 (A) synthetase can be efficiently expressed in insect cells using the baculovirus system. The recombinant protein was purified to apparent homogeneity, and its enzymatic activity 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 studying the properties of two previously reported mutant proteins. Both of these mutants, when produced in bacteria, are enzymatically inactive, although similarly produced wild-type protein is active. Unexpectedly, when expressed in insect cells, both mutant proteins were enzymatically as active as the wild-type protein. These results suggest that in the eukaryotic expression system described here, the mutant proteins can undergo appropriate modifications or folding that is required for attaining an enzymatically active conformation.
Bandyopadhyay
S
S
Department of Molecular Biology, Lerner Research Institute, Cleveland Clinic Foundation, Ohio 44195, USA.
Ghosh
A
A
Sarkar
S N
SN
Sen
G C
GC
D015088:2',5'-Oligoadenylate Synthetase / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001120:Arginine / Q000235:genetics; D016367:Baculoviridae / Q000201:enzymology / Q000235:genetics*; D005822:Genetic Vectors / Q000138:chemical synthesis; D005973:Glutamine / Q000235:genetics; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D008239:Lysine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D018411:Spodoptera / Q000201:enzymology / Q000235:genetics
D007527:Isoenzymes; D011994:Recombinant Proteins; D005973:Glutamine; D001120:Arginine; D011392:Proline; D015088:2',5'-Oligoadenylate Synthetase; D008239:Lysine