A highly thermostable, homodimeric single-stranded DNA-binding protein from Deinococcus radiopugnans.
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.
Filipkowski
Paweł
P
Department of Microbiology, Gdańsk University of Technology, ul. Narutowicza 11/12, 80-952 Gdańsk, Poland.
Koziatek
Magdalena
M
Kur
Józef
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D034301:Deinococcus / Q000737:chemistry* / Q000235:genetics; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011489:Protein Denaturation; D017510:Protein Folding; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C476430:SSB protein, E coli