title: [Characterization of recombinant single-stranded DNA-binding protein from Escherichia coli and its application in accurate pyrosequencing].
abstract: We expressed recombinant single-stranded DNA-binding protein (r-SSBP) from Escherichia coli with the molecular weight of 24-kDa by using genetic engineering strategy, and demonstrated the single-stranded DNA (ssDNA)-binding activity of r-SSBP by electrophoretic mobility shift assay (EMSA). To further characterize r-SSBP, we studied the effects of r-SSBP on melting temperature (T(m)) of DNA. The results showed that r-SSBP could bind to ssDNA, and lower the T(m) of DNA, especially for single-base mismatched DNA. Therefore, r-SSBP significantly increased the T(m) difference between single-base mismatched DNA and perfect matched DNA. These results are very beneficial for single-nucleotide polymorphism detection. Moreover, we applied r-SSBP in high sensitive pyrosequencing system developed by our group. The results suggest that the r-SSBP decreased non-specific signals, corrected the proportion of signal peak height and improved the performance of pyrosequencing.
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Jianping
authors.initials: J
authors.lastname: Wang
authors.forename: Bingjie
authors.initials: B
authors.lastname: Zou
authors.forename: Zhiyao
authors.initials: Z
authors.lastname: Chen
authors.forename: Yinjiao
authors.initials: Y
authors.lastname: Ma
authors.forename: Shu
authors.initials: S
authors.lastname: Xu
authors.forename: Guohua
authors.initials: G
authors.lastname: Zhou
mesh_terms: D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D011756:Diphosphates / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D004268:DNA-Binding Proteins; D011756:Diphosphates; D011994:Recombinant Proteins