Cloning, High Expression of Single-Stranded DNA-Binding Protein and Its Interaction with ssDNA
10.3969/j.issn.1671-8135.2007.04.003
- VernacularTitle:SSB蛋白的高效表达及其与ssDNA的相互作用
- Author:
Hui-Li QIAO
1
;
Yuan-Yuan CHEN
;
Zhen-Zhong WEN
;
Li-Jun BI
;
Yun-Chao KAN
Author Information
1. 南阳师范学院
- Keywords:
SSB High expression Interaction Kinetic assay
- From:
China Biotechnology
2007;27(4):12-17
- CountryChina
- Language:Chinese
-
Abstract:
E.coli single-stranded DNA-binding protein (SSB) plays an important role in replication, recombination and repair of DNA and is thus crucial for the survival of the bacteria.We described a high expression and efficient purification scheme and kinetic assay of interaction with its substrate, single-stranded DNA (ssDNA). A ssb gene (537 bp) for encoding SSB was obtained by PCR amplification from E.coli K-12 genome. The expression vector of the fusion protein SSB was constructed by attaching ssb gene to pQE30. SSB fusion protein was expressed in M15 E.coli strain induced by IPTG. SDS-PAGE analysis revealed that the expected protein with a molecular weight 20.6kDa was soluble and amounted to about 30% of the total bacterial protein. SSB protein was purified by immobilized metal (Ni2+) chelation affinity chromatography and the purity was about 90%. The resulting SSB protein was a correctly folded tetramer analyzed by gel filtration. It could bind ssDNA with equilibrium dissociation constant (KD) of 4.79×10-7 mol/L as determined by surface plasmon resonance.
