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Structural features of the initiator of replication protein RepB encoded by the promiscuous plasmid pMV158

José A Ruiz-Masó1, Consuelo López-Zumel, Margarita Menéndez

  • 1Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas, Velázquez, 144, E-28006 Madrid, Spain.

Biochimica Et Biophysica Acta
|January 17, 2004
PubMed

Insights

Researchers developed a new method to overproduce and purify the RepB replication initiator protein. Purified RepB functions as a hexamer and undergoes temperature-induced conformational changes, explaining its high optimal activity temperature.

Area of Science:

  • Molecular Biology
  • Protein Biochemistry

Background:

  • The rolling circle (RC) replicating plasmid pMV158 encodes the RepB initiator protein, essential for DNA replication.
  • RepB exhibits topoisomerase I-like activity, relaxing supercoiled DNA.

Purpose of the Study:

  • To develop an efficient method for overproduction, scaling-up, and purification of the RepB protein.
  • To characterize the oligomeric state and structural properties of purified RepB.

Main Methods:

  • Development of a novel vector and purification protocol for RepB overproduction.
  • Analytical ultracentrifugation to determine the oligomeric state of RepB in solution.
  • Circular dichroism (CD) spectroscopy to assess protein secondary structure and thermal stability.

Main Results:

  • RepB was successfully overproduced and purified, existing as a hexamer in solution.
  • CD analysis revealed RepB secondary structure content (approx. 33% alpha-helix, 20% beta-strand).
  • Temperature-induced conformational changes were observed above 35°C, indicated by irreversible spectral shifts.

Conclusions:

  • The developed method enables efficient production and purification of functional RepB protein.
  • The hexameric state and temperature-dependent conformational changes of RepB are crucial for its catalytic activity at elevated temperatures.

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