What question did this study set out to answer?

Express and purify poliovirus RNA polymerase in Escherichia coli to evaluate its catalytic behavior and template copying mechanisms in vitro.

January 1, 1989Journal of VirologyOpen Access

Purification and properties of poliovirus RNA polymerase expressed in Escherichia coli

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Population

Escherichia coli expressing a cDNA clone encoding the RNA polymerase of poliovirus

Design

Preclinical

Authors

No immediate clinical implications; leaves open further mechanistic and antiviral studies of picornavirus polymerases.

Express and purify poliovirus RNA polymerase in Escherichia coli to evaluate its catalytic behavior and template copying mechanisms in vitro.
Cloned poliovirus RNA polymerase cDNA with an engineered N-terminal methionine behind a T7 bacteriophage promoter for expression in Escherichia coli.
Purified the recombinant enzyme to near homogeneity and generated specific polyclonal antibodies against it.
Evaluated enzymatic RNA synthesis and primer dependence using poliovirus and globin RNA templates alongside [alpha-32P]UTP labeling.
Demonstrated poly(A)-dependent oligo(U)-primed poly(U) polymerase activity and template-directed RNA polymerase activity.
Synthesized full-length copies of poliovirus and globin RNA templates in the presence of an oligo(U) primer.
Produced RNA molecules up to twice the template length without an added primer and incorporated [alpha-32P]UTP directly into template RNA.

Population

Escherichia coli expressing a cDNA clone encoding the RNA polymerase of poliovirus

Intervention

Expression and purification of recombinant poliovirus RNA polymerase

Outcome

Enzyme activity (poly(A)-dependent oligo(U)-primed poly(U) polymerase activity and RNA polymerase activity)surrogate

Recombinant poliovirus RNA polymerase expressed in E. coli demonstrates functional polymerase activity in vitro.

Plotch et al. (1989) studied this question.

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