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Open Access Research article

Characterization of exceptionally thermostable single-stranded DNA-binding proteins from Thermotoga maritima and Thermotoga neapolitana

Marcin Olszewski1*, Anna Grot1, Marek Wojciechowski2, Marta Nowak1, Małgorzata Mickiewicz1 and Józef Kur1

Author Affiliations

1 Gdańsk University of Technology, Department of Microbiology, ul. Narutowicza 11/12, 80-233 Gdańsk, Poland

2 Gdańsk University of Technology, Department of Pharmaceutical Technology and Biochemistry, ul. Narutowicza 11/12, 80-233 Gdańsk, Poland

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BMC Microbiology 2010, 10:260  doi:10.1186/1471-2180-10-260

Published: 15 October 2010

Abstract

Background

In recent years, there has been an increasing interest in SSBs because they find numerous applications in diverse molecular biology and analytical methods.

Results

We report the characterization of single-stranded DNA binding proteins (SSBs) from the thermophilic bacteria Thermotoga maritima (TmaSSB) and Thermotoga neapolitana (TneSSB). They are the smallest known bacterial SSB proteins, consisting of 141 and 142 amino acid residues with a calculated molecular mass of 16.30 and 16.58 kDa, respectively. The similarity between amino acid sequences of these proteins is very high: 90% identity and 95% similarity. Surprisingly, both TmaSSB and TneSSB possess a quite low sequence similarity to Escherichia coli SSB (36 and 35% identity, 55 and 56% similarity, respectively). They are functional as homotetramers containing one single-stranded DNA binding domain (OB-fold) in each monomer. Agarose mobility assays indicated that the ssDNA-binding site for both proteins is salt independent, and fluorescence spectroscopy resulted in a size of 68 ± 2 nucleotides. The half-lives of TmaSSB and TneSSB were 10 h and 12 h at 100°C, respectively. When analysed by differential scanning microcalorimetry (DSC) the melting temperature (Tm) was 109.3°C and 112.5°C for TmaSSB and TneSSB, respectively.

Conclusion

The results showed that TmaSSB and TneSSB are the most thermostable SSB proteins identified to date, offering an attractive alternative to TaqSSB and TthSSB in molecular biology applications, especially with using high temperature e. g. polymerase chain reaction (PCR).