title: Novel thermostable ssDNA-binding proteins from Thermus thermophilus and T. aquaticus-expression and purification.
abstract: Single-stranded DNA-binding proteins (SSBs) play essential roles in DNA replication, recombination, and repair in bacteria, archaea, and eukarya. We report here the identification, expression, and purification of the SSB-like proteins of the thermophilic bacteria Thermus thermophilus and T. aquaticus. The nucleotide (nt) sequence revealed that T. thermophilus SSB (TthSSB) and T. aquaticus (TaqSSB) consist of 264 and 266 amino acids, respectively, and have a molecular weight of 29.87 and 30.03kDa, respectively. The homology between these protein, is very high-82% identity and 90% similarity. They are the largest known prokaryotic SSB proteins. TthSSB and TaqSSB monomers have two putative ssDNA-binding sequences: N-terminal (located in the region from amino acids 1 to 123) and C-terminal (located between amino acids 124 and 264 or 266 in TthSSB and TaqSSB, respectively). PCR-derived DNA fragment containing the complete structural gene for TthSSB or TaqSSB protein was cloned into an expression vector. The clones expressing SSB-like proteins were selected and cloned DNA fragments were verified to be authentic by sequencing several clones. The purification was carried out using reduction of contamination by the host protein with heat treatment, followed by QAE-cellulose and ssDNA-cellulose column chromatography. We found our expression and purification system to be quite convenient and efficient, and will use it for production of thermostable SSB-proteins for crystallography study. We have applied the use of TthSSB and TaqSSB protein to increase the amplification efficiency with a number of diverse templates. The use of SSB protein may prove to be generally applicable in improving the PCR efficiency.
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D015870:Gene Expression; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D017385:Sequence Homology; D013696:Temperature; D013824:Thermus / Q000235:genetics*; D016963:Thermus thermophilus / Q000235:genetics*
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins