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|標題:||Synthesis in Escherichia coli of avian reovirus core protein sigma A and its dsRNA-binding activity||作者:||Yin, H.S.
|關鍵字:||double-stranded-rna;structural proteins;coding assignment;genome;segments;strain s1133;virus;gene;polypeptides;sequences;cloning||Project:||Virology||期刊/報告no：:||Virology, Volume 266, Issue 1, Page(s) 33-41.||摘要:||
The genome segment S2 of avian reovirus (ARV) S1133 was cloned and sequenced. The entire S2 nucleotide sequence is 1325 bp long with one long open reading frame that encodes a protein of 415 amino acids, corresponding to sigma A, a major core protein of ARV. S2 possesses a pentanucleotide, TCATC, at the 3'-terminus of its plus strand, common to other known genome segments of ARV and to 10 genome segments of mammalian reovirus. Amino acid sequence analysis revealed that sigma A contains a carboxy-terminal region (one-fourth of the protein) mat is formed from alpha-helices and beta-turns, and the remainder (three-fourths of the protein) is formed predominantly from beta-strands and beta-turns. Analysis of binding activity to poly(rl)-poly(rC)-agarose suggested that ARV protein A present in total virus-infected chicken embryo fibroblasts (CEF) had dsRNA-binding activity. To further characterize the binding activity, protein sigma A was subsequently expressed in Escherichia coli BL21(DE3) cells as a fusion protein and isolated by metal chef ate affinity chromatography. The expressed protein e sigma A was further purified through a Superdex 75 HR 10/30 column after digestion of me purified fusion peptide with enterokinase. The expressed protein e sigma A has the same molecular weight as virion protein sigma A purified from ARV-infected CEF and is indistinguishable from virion protein sigma A by immunoblot analysis. The e sigma A binds cooperatively alpha P-32-labeled dsRNA probe produced by run-off transcription of clone pGEM-3Zf(+)S4. The binding reaction is blocked by homologous ARV dsRNA or heterologous infectious bursal disease virus dsRNA and poly(rl)-poly(rC), but not by salmon sperm DNA. The results indicate that the expressed protein e sigma A has dsRNA-binding activity similar to that of sigma A obtained from infected cells, and its binding is sequence-independent. (C) 2000 Academic Press.
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