Characterization Of Recombinant Escherichia Coli Single-Strand Binding Protein And Its Application In Pyrosequencing

We expressed recombinant single-strand 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 the r-SSBP by electrophoretic mobility shift assay (EMSA). To further characterize the r-SSBP, we studied the effects of the r-SSBP on melting temperature (T-m) of DNA. The results showed that the r-SSBP can bind to ssDNA and can lower the T-m of DNA, especially for single-base mismatched DNA. Therefore, the r-SSBP can significantly increase 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 the r-SSBP in high sensitive pyrosequencing system developed by our group. The results suggested that the r-SSBP can decrease unspecific signals, correct the proportion of signal peak height, and improve the performance of pyrosequencing. This research laid the foundation for the development of high sensitive pyrosequencing regents.