Expression and characterization of recombinant 2',5'-oligoadenylate synthetase from the marine sponge Geodia cydonium.
2',5'-oligoadenylate (2-5A) synthetases are known as components of the interferon-induced cellular defence mechanism in mammals. The existence of 2-5A synthetases in the evolutionarily lowest multicellular animals, the marine sponges, has been demonstrated and the respective candidate genes from Geodia cydonium and Suberites domuncula have been identified. In the present study, the putative 2-5A synthetase cDNA from G. cydonium was expressed in an Escherichia coli expression system to characterize the enzymatic activity of the recombinant polypeptide. Our studies reveal that, unlike the porcine recombinant 2-5A synthetase, the sponge recombinant protein associates strongly with RNA from E. coli, forming a heterogeneous set of complexes. No complete dissociation of the complex occurs during purification of the recombinant protein and the RNA constituent is partially protected from RNase degradation. We demonstrate that the sponge recombinant 2-5A synthetase in complex with E. coli RNA catalyzes the synthesis of 2',5'-phosphodiester-linked 5'-triphosphorylated oligoadenylates from ATP, although with a low specific activity. Poly(I).poly(C), an efficient artificial activator of the mammalian 2-5A synthetases, has only a minimal effect (an approximate two-fold increase) on the sponge recombinant 2-5A synthetase/bacterial RNA complex activity.
Päri
Mailis
M
Department of Gene Technology, Tallinn University of Technology, Estonia.
Kuusksalu
Anne
A
Lopp
Annika
A
Reintamm
Tõnu
T
Justesen
Just
J
Kelve
Merike
M
D015088:2',5'-Oligoadenylate Synthetase / Q000737:chemistry*; D000818:Animals; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D019143:Evolution, Molecular; D005786:Gene Expression Regulation; D006639:Histidine / Q000737:chemistry; D011161:Porifera; D012313:RNA / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
D018076:DNA, Complementary; D011994:Recombinant Proteins; D006639:Histidine; D012313:RNA; D015088:2',5'-Oligoadenylate Synthetase