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Direct expression of a modified foot-and-mouth-disease virus RNA-polymerase in Escherichia coli

A. Tanuri^I,II; P.J. Vieira^I; R.C. Olascoaga^I

^ICentro Panamericano de Febre Aftosa, Duque de Caxias, 20000 Rio de Janeiro, Brasil
^IIPresent address: Laboratório de Fisiologia Celular, Instituto de Biofísica Carlos Chagas Filho, Cidade Universitária, UFRJ, Caixa Postal 68047, 21941 Rio de Janeiro, RI, Brasil. Send correspondence to A.

ABSTRACT

Foot-and-mouth disease virus (FMDV) RNA-polymerase (RNA-pol) was expressed at high levels in an Escherichia coli strain, in a controllable fashion. An expression vector was constructed by cloning the cDNA of the FMDV RNA-pol, from its twenty-first codon to its 3'translational termination signal, into plasmid pPL, under the control of the PL promoter. Expression levels up to 10% of the total cellular protein were achieved upon temperature induction, and a protein of the expected molecular weight (50 kDa) could be visualized by SDS-PAGE, which cross-reacted with rabbit anti-RNA-pol and FMDconvalescent bovine sera in Western blotting. Mouse hyperimmune antiserum to the recombinant RNA-pol was able to inhibit the poly (U) polymerase activity of the natural RNA-polymerase. Such a direct expression of the RNA-pol in E. coli will certainly help the analysis of the activity of this enzyme and might also make possible the development of a more efficient and safe diagnostic kit for FMDV.

Keywords: Modified foot; Mouth-disease; Virus; RNA-polymerase; Escherichia coli.

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