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Updated: Jul 18, 2026

An Assay for Quantifying Protein-RNA Binding in Bacteria
Published on: June 12, 2019
Identification, cloning, expression, and characterization of a highly thermostable single-stranded-DNA-binding
Paweł Filipkowski1, Anna Duraj-Thatte, Józef Kur
1Gdańsk University of Technology, Chemical Faculty, Department of Microbiology, ul. Narutowicza 11/12, 80-952 Gdańsk, Poland.
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 degrees C, with half-lives of 50 min at 95 degrees C, 40 min at 100 degrees C and 35 min at 105 degrees 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.
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