Characterization of avian reovirus non structural protein sigmaNS synthesized in Escherichia coli.
The coding region of avian reovirus S1133 genomic segment S4, encoding the non structural protein sigmaNS, was inserted into expression vector pET28a and the protein was expressed in Escherichia coli BL21(DE3) as a fusion protein containing a C-terminal peptide with six tandem histidines (His-tag). The expressed protein (esigmaNS) consistent with the expected molecular size of the avian reovirus protein sigmaNS synthesized in infected cells was readily purified by His-Bind Resin. The esigmaNS was further confirmed to be indistinguishable from viral sigmaNS by immunoblot analysis. The esigmaNS binds 32P-labeled ssRNA probe produced by run-off transcription of clone pGEM-3Zf(+)S4. The binding activity is blocked by heterologous yeast rRNA, but not by homologous avian reovirus dsRNA and heterologous infectious bursal disease virus dsRNA and salmon sperm dsDNA. Therefore, the ssRNA-binding activity of the expressed protein sigmaNS is non sequence-specific, similar to that previously described for viral sigmaNS purified from avian reovirus infected cell extracts. In addition, the recent data also show that the optimal salt (NaCl) concentration and pH for its binding are 100-150 mM and 7.0, respectively, in terms of the UV cross-linking and RNase A treatment of the reaction mixtures prior to the denaturing gel analysis.
Yin
H S
HS
Department of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan.
Lee
L H
LH
D000818:Animals; D002478:Cells, Cultured / Q000821:virology; D002642:Chick Embryo; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005347:Fibroblasts / Q000821:virology; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D018165:Orthoreovirus / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012965:Sodium Chloride; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C031657:sigma NS protein, Reovirus; D012965:Sodium Chloride; D012313:RNA