Synthesis of the major inner capsid protein VP6 of the human rotavirus Wa in Escherichia coli.
The gene for the major inner capsid protein VP6 of human rotavirus strain Wa has been cloned and placed into a bacterial expression vector under the control of the inducible hybrid trp-lac (tac) promoter. Recombinant VP6 was produced at low levels in a cell-free Escherichia coli transcription-translation system programmed with this expression plasmid. The yield of VP6 synthesized in the extract could be increased several-fold by introduction of point mutations upstream and downstream from the start codon. Upon induction with IPTG, E. coli JM105 cells harboring the mutated expression plasmid produced VP6 as shown by immunoblotting of proteins from bacterial lysates with anti-Wa antiserum. Recombinant VP6 appeared to inhibit the growth of E. coli and did not accumulate in the cells to high levels. Conformational analysis with a monoclonal antibody suggested that bacterially produced VP6 adopted an oligomeric structure characteristic for native VP6.
Ernst
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Department of Clinical Virology, James N. Gamble Institute of Medical Research, Cincinnati, OH 45219.
Stroup
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005814:Genes, Viral; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis; D012401:Rotavirus / Q000235:genetics*; D014158:Transcription, Genetic
D015345:Oligonucleotide Probes; D011994:Recombinant Proteins