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. Ho wever, high-level expression of 3D(pol) in E. coli yields enzyme with a het erogeneous amino terminus. To preclude this problem, we developed a new exp ression system for 3D(pol). This system exploits the observation that prote ins 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 expresse d in the presence of a ubiquitin-specific, carboxy-terminal protease. By us ing this system, authentic 3D(pol) can be obtained in yields of 30-60 mg pe r liter of culture. While addition of a single glycine, alanine, serine, or valine to the amino terminus of 3D(pol) produced derivatives with a specif ic activity reduced by at least 25-fold relative to wild-type enzyme, addit ion of a methionine to the amino terminus resulted in some processing to yi eld enzyme with a glycine amino terminus, Addition of a hexahistidine tag t o the carboxyl terminus of 3D(pol) had no deleterious effect on the activit y of the enzyme. The utility of this expression system for production of ot her viral polymerases and accessory proteins is discussed. (C) 1999 Academi c Press.