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A highly thermostable, homodimeric single-stranded DNA-binding protein from Deinococcus radiopugnans
Abstract
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°C, 60 min at 95°C and 30 min at 100°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°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.
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Keywords
Details
- Category:
- Articles
- Type:
- artykuł w czasopiśmie wyróżnionym w JCR
- Published in:
-
EXTREMOPHILES
no. 10,
edition 6,
pages 607 - 614,
ISSN: 1431-0651 - Language:
- English
- Publication year:
- 2006
- Bibliographic description:
- Filipkowski P., Koziatek M., Kur J.: A highly thermostable, homodimeric single-stranded DNA-binding protein from Deinococcus radiopugnans// EXTREMOPHILES. -Vol. 10, iss. 6 (2006), s.607-614
- DOI:
- Digital Object Identifier (open in new tab) 10.1007/s00792-006-0011-8
- Verified by:
- Gdańsk University of Technology
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