High-level synthesis of cowpea mosaic virus RNA polymerase and protease in Escherichia coli.
An expression system for the production of polymerase proteins of cowpea mosaic virus (CPMV) in Escherichia coli cells is described. High-level synthesis of proteins containing protease and polymerase moieties (110-kDa protein) and polymerase alone (87-kDa protein) were obtained from cells containing different plasmid constructions. Precursor and processed forms of CPMV proteins were detected by immunoblotting with antisera directed against 170-kDa precursor polyprotein and 24-kDa viral protease. Crude lysates and supernatant fractions of the lysates from E. coli cells harboring the various plasmid constructions were analysed for poly(A)-oligo(U) polymerase activity and found to be negative for CPMV activity under conditions where similar expression systems for the production of poliovirus RNA polymerase activity were positive. Thus, conditions for CPMV RNA replication may indeed be different from those for poliovirus even though the genomic organization of these viruses is similar.
Richards
O C
OC
Department of Molecular Biology, Agricultural University, Wageningen, The Netherlands.
Eggen
R
R
Goldbach
R
R
van Kammen
A
A
D004274:DNA, Recombinant; D012321:DNA-Directed RNA Polymerases / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015151:Immunoblotting; D009029:Mosaic Viruses / Q000201:enzymology / Q000235:genetics*; D010447:Peptide Hydrolases / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D010957:Plasmids; D017955:Poliovirus / Q000235:genetics; D012995:Solubility
D004274:DNA, Recombinant; D012321:DNA-Directed RNA Polymerases; D010447:Peptide Hydrolases