Single-stranded DNA-binding proteins (SSBs) play essential roles in DNA replication, recombination, and repair in bacteria, archaea and eukarya. The SSBs share a common core ssDNA-binding domain with a conserved OB (oligonucleotide/oligosaccharide binding) fold. This ssDNA-binding domain was presumably present in the common ancestor to all three major branches of life. In recent years, there has been an increasing interest in SSBs because they are useful for molecular biology methods and for analytical purposes. In this review, we concentrate on recent advances in the discovery of new sources of SSBs as well as certain aspects of their applications in analytical sciences.

Two recombinant trehalose synthases from Deinococcus geothermalis (DSMZ 11300) were compared. A significant influence of the artificial polyhistidine tag was observed in protein constitution. The recombinant trehalose synthase from D. geothermalis with His₆-tag has a higher Km value of 254 mM, in comparison with the wild-type trehalose synthase (Km 170 mM), and displayed a lower activity of maltose conversion when compared to the wild type. Moreover, differences in properties like temperature, pH, thermal- and pH-stability were observed. Presence of the histidine tag caused a decrease of thermal resistance in case of trehalose synthase with His₆-tag. These data confirmed a suggestion that the introduction of the histidine domain produces in some seldom cases undesirable changes in the protein.

A trehalose synthase gene from Deinococcus radiodurans (DSMZ 20539) containing 1659 bp reading frame encoding 552 amino acids was amplified using PCR. The gene was finally ligated into pET30Ek/LIC vector and expressed after isopropyl β-d-thiogalactopyranoside induction in Escherichia coli (DE3) Rosetta pLysS. The recombinant trehalose synthase (DraTreS) containing a His 6 -tag at the C-terminus was purified by metal affinity chromatography and characterized. The expressed enzyme is a homodimer with molecular mass of 126.9 kDa and exhibits the highest activity of 11.35 U/mg at pH 7.6 and at 30oC. DraTreS activity was almost unchanged after 2 h preincubation at 45oC and pH 7.6, and retained about 56% of maximal value after 8 h incubation at 50oC. The DraTreS was strongly inhibited by Cu 2+ , Hg 2+ , Zn 2+ , Al 3+ and 10 mM Tris. The K m value of maltose conversion was 290.7 mM.

To study the biochemical properties of single-stranded DNA-binding (SSB) protein from Deinococcus geothermalis (DgeSSB), we have cloned the ssb gene obtained by PCR and developed an overexpression system. The gene consists of an open reading frame of 900 nucleotides encoding a protein of 300 amino acids with a calculated molecular weight of 32.45 kDa. The amino acid sequence exhibits 43, 44 and 75% identity with Thermus aquaticus, Thermus thermophilus and Deinococcus radiodurans SSBs, respectively. We show that DgeSSB is similar to Thermus/Deinococcus SSB in its biochemical properties. DgeSSB includes two oligonucleotide/oligosaccharide-binding folds per monomer and functions as a homodimer. In fluorescence titrations with poly(dT), DgeSSB bound about 30 nt independent of the salt concentration, and the fluorescence was quenched by about 65%. In a complementation assay in Escherichia coli, DgeSSB took over the in vivo function of EcoSSB. DgeSSB is thermostable with half-lives of 50 min at 70 degrees C and 5 min at 90 degrees C. Hence, DgeSSB offers an attractive alternative for TaqSSB and TthSSB in their applications for molecular biology methods and for analytical purposes.

We report the identification and characterization of the single-stranded DNA-binding protein (SSB) from the mesophile and highly radiation-resistant Deinococcus radiopugnans (DrpSSB). PCR-derived DNA fragment containing the complete structural gene for DrpSSB protein was cloned and expressed in Escherichia coli. The gene consisting of an open reading frame of 900 nucleotides encodes a protein of 300 amino acids with a calculated molecular weight of 32.45 kDa and pI 5.34. The amino acids sequence exhibits 43, 44, 79 and 18% identity with Thermus aquaticus, Thermus thermophilus, Deinococcus radiodurans and E. coli SSBs, respectively. The DrpSSB includes two OB folds per monomer and functions as a homodimer. In fluorescence titrations with poly(dT), DrpSSB bound 24-31 nt depending on the salt concentration, and fluorescence was quenched by about 80%. In a complementation assay in E. coli, DrpSSB took over the in vivo function of EcoSSB. The half-lives of DrpSSB were 120 min at 90 degrees C, 60 min at 95 degrees C and 30 min at 100 degrees C. These results were surprising in the context of half-life of SSB from thermophilic T. aquaticus, which has only 30 s of half-life at 95 degrees C. DrpSSB 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 analytical purposes.