Characterization of recombinant single-stranded DNA-binding protein from Escherichia coli and its application in accurate pyrosequencing.
- Author:
Jianping WANG
1
;
Bingjie ZOU
;
Zhiyao CHEN
;
Yinjiao MA
;
Shu XU
;
Guohua ZHOU
Author Information
1. School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China.
- Publication Type:Journal Article
- MeSH:
DNA-Binding Proteins;
biosynthesis;
genetics;
Diphosphates;
metabolism;
Escherichia coli;
genetics;
metabolism;
Genetic Vectors;
Recombinant Proteins;
biosynthesis;
genetics;
Sequence Analysis, DNA;
methods
- From:
Chinese Journal of Biotechnology
2011;27(10):1513-1520
- CountryChina
- Language:Chinese
-
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.
