Identification, cloning, expression, and characterization of a highly thermostable single-stranded-DNA-binding protein (SSB) from Deinococcus murrayi

We report identification and characterization of SSB-like protein from Deinococcus murrayi (DmuSSB). PCR-derived DNA fragment containing the complete structural gene for DmuSSB was cloned and expressed in Escherichia coli. The gene consisted of an open reading frame of 826 nucleotides encoding a protein of 276 amino acid residues with a calculated molecular weight of 30.14 kDa. DmuSSB includes two OB folds per monomer and functions as a homodimer. In fluorescence titrations with poly(dT) DmuSSB bound 27–32 nt depending on the salt concentration, and fluorescence was quenched by about 62%. In a complementation assay in E. coli, DmuSSB took over the in vivo function of EcoSSB. DmuSSB maintained 100% activity after 120 min incubation at 80 °C, with half-lives of 50 min at 95 °C, 40 min at 100 °C and 35 min at 105 °C. DmuSSB is the most thermostable SSB-like protein identified to date, offering an attractive alternative for TaqSSB and TthSSB in their applications for molecular biology methods and for analytical purposes.