Purification and characterization of the Thermus thermophilus HB8 RecX protein

Koutaro Jimbo, Jin Inoue, Tokiha Masuda, Takehiko Shibata, Tsutomu Mikawa

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)

Abstract

The RecA protein plays a central role in homologous recombination by promoting strand exchange between ssDNA and homologous dsDNA. Since RecA alone can advance this reaction in vitro, it is widely used in gene manipulation techniques. The RecX protein downregulates the function of RecA, indicating that it could be used as an inhibitor to control the activities of RecA in vitro. In this study, the RecX protein of the hyper-thermophilic bacterium Thermus thermophilus (ttRecX) was over-expressed in Escherichia coli and purified by heat treatment and several column chromatography steps. Size-exclusion chromatography indicated that purified ttRecX exists as a monomer in solution. Circular dichroism measurements indicated that the α-helical content of ttRecX is 54% and that it is stable up to 80 °C at neutral pH. In addition, ttRecX inhibited the DNA-dependent ATPase activity of the T. thermophilus RecA protein (ttRecA). The stable ttRecX may be applicable for variety of techniques using the ttRecA reaction.

Original languageEnglish
Pages (from-to)320-323
Number of pages4
JournalProtein Expression and Purification
Volume51
Issue number2
DOIs
Publication statusPublished - 2007 Feb
Externally publishedYes

Keywords

  • RecA
  • Recombination
  • Thermostable
  • Thermus thermophilus

ASJC Scopus subject areas

  • Biotechnology

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