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Facile Production and Rapid Purification of Functional Recombinant Qβ Replicase Heterotetramer Complex

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Abstract

We describe an improved method for the production of recombinant Qβ replicase heterotetramer. The successful expression of the soluble Qβ RNA polymerase complex depends on the EF-Ts and EF-Tu subunits being co-expressed prior to β-subunit expression. Efficient co-expression requires two different inducible operons to co-ordinate the expression of the heterotrimer. The complete heterotetramer enzyme complex is achieved by production of the recombinant S1-subunit of Qβ replicase in a separate host. This approach represents a facile way for producing and purifying large amounts of soluble and active recombinant Qβ replicase tetramer without the necessity of a His-tag for purification.

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Acknowledgments

We thank Dr. R. Anitori for providing plasmid pJLA602-Qβ. This work was funded by the Environmental Biotechnology Cooperative Research Centre (EBCRC).

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Authors and Affiliations

  1. Department of Chemistry and Biomolecular Sciences, and Environmental Biotechnology CRC, Macquarie University, North Ryde, 2109, Sydney, NSW, Australia

    Karthikeyan Gunasekaran, Peter L. Bergquist & Anwar Sunna

  2. Department of Molecular Medicine & Pathology, Medical School, University of Auckland, Auckland, New Zealand

    Peter L. Bergquist

Authors
  1. Karthikeyan Gunasekaran
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  2. Peter L. Bergquist
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  3. Anwar Sunna
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Correspondence to Anwar Sunna.

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Gunasekaran, K., Bergquist, P.L. & Sunna, A. Facile Production and Rapid Purification of Functional Recombinant Qβ Replicase Heterotetramer Complex. Appl Biochem Biotechnol 169, 651–659 (2013). https://doi.org/10.1007/s12010-012-0018-9

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