Identification, cloning, expression, and characterization of a highly thermostable single-stranded-DNA-binding protein (SSB) from Deinococcus murrayi.
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.
Filipkowski
Paweł
P
Gdańsk University of Technology, Chemical Faculty, Department of Microbiology, ul. Narutowicza 11/12, 80-952 Gdańsk, Poland.
Duraj-Thatte
Anna
A
Kur
Józef
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D034301:Deinococcus / Q000235:genetics*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006207:Half-Life; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D014170:Transformation, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins