中国生物工程杂志

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. In this report, we described a high expression and efficient purification scheme and kinetic assay on 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 induced with IPTG. SDS-PAGE revealed that the expected protein with a molecular weight 20.6kD 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 with an apparent binding to ssDNA with equilibrium dissociation constant (KD) of 4.79×10-7 M as determined by gel filtration and surface plasmon resonance.