Characterization of avian reovirus non structural protein sigma NS synthesized in Escherichia coli

The coding region of avian reovirus S1133 genomic segment S4, encoding the non structural protein sigma NS, was inserted into expression vector pET28a and the protein was expressed in Escherichia coli BL21(DE3) as a fusion pr otein containing a C-terminal peptide with six tandem histidines (His-tag). The expressed protein (e sigma NS) consistent with the expected molecular size of the avian reovirus protein sigma NS synthesized in infected cells w as readily purified by His-Bind Resin. The e sigma NS was further confirmed to be indistinguishable from viral sigma NS by immunoblot analysis. The e sigma NS binds P-32-labeled ssRNA probe produced by run-off transcription o f clone pGEM-3Zf(+)S4. The binding activity is blocked by heterologous yeas t rRNA, but not by homologous avian reovirus dsRNA and heterologous infecti ous bursal disease virus dsRNA and salmon sperm dsDNA. Therefore, the ssRNA -binding activity of the expressed protein sigma NS is non sequence-specifi c, similar to that previously described for viral sigma NS purified from av ian 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. (C ) 2000 Elsevier Science B.V. All rights reserved.