Production of "authentic" poliovirus RNA-dependent RNA polymerase (3D(pol)) by ubiquitin-protease-mediated cleavage in Escherichia coli.
The first amino acid of "authentic" poliovirus RNA-dependent RNA polymerase, 3D(pol), is a glycine. As a result, production of 3D(pol) in Escherichia coli requires addition of an initiation codon; thus, a formylmethionine is added to the amino terminus. The formylmethionine should be removed by the combined action of a cellular deformylase and methionine aminopeptidase. However, high-level expression of 3D(pol) in E. coli yields enzyme with a heterogeneous amino terminus. To preclude this problem, we developed a new expression system for 3D(pol). This system exploits the observation that proteins fused to the carboxyl terminus of ubiquitin can be processed in E. coli to produce proteins with any amino acid as the first residue when expressed in the presence of a ubiquitin-specific, carboxy-terminal protease. By using this system, authentic 3D(pol) can be obtained in yields of 30-60 mg per liter of culture. While addition of a single glycine, alanine, serine, or valine to the amino terminus of 3D(pol) produced derivatives with a specific activity reduced by at least 25-fold relative to wild-type enzyme, addition of a methionine to the amino terminus resulted in some processing to yield enzyme with a glycine amino terminus. Addition of a hexahistidine tag to the carboxyl terminus of 3D(pol) had no deleterious effect on the activity of the enzyme. The utility of this expression system for production of other viral polymerases and accessory proteins is discussed.
Gohara
D W
DW
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Ha
C S
CS
Kumar
S
S
Ghosh
B
B
Arnold
J J
JJ
Wisniewski
T J
TJ
Cameron
C E
CE
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D005814:Genes, Viral; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D017955:Poliovirus / Q000201:enzymology* / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D012324:RNA-Dependent RNA Polymerase / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D014452:Ubiquitins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; C090443:polymerase 3Dpol, poliovirus; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases; D010450:Endopeptidases