Article ID Journal Published Year Pages File Type
2022016 Protein Expression and Purification 2007 8 Pages PDF
Abstract

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.

Related Topics
Life Sciences Biochemistry, Genetics and Molecular Biology Biochemistry
Authors
, , ,