中文摘要：

大肠杆菌单链结合蛋白SSB在DNA复制、重组和修复中起着重要作用。为研究单链结合蛋白SSB的体外生物功能构建了融合蛋白SSB的表达载体并使其高效表达及易于纯化。ssb基因片段是以E.coliK-12基因组为模板经PCR扩增获得,并通过基因的体外拼接成功构建了表达载体pQE30-ssb。重组菌株M15/pQE30-ssb经过IPTG的诱导表达了蛋白SSB。收集菌体细胞、超声波破碎后离心取上清进行SDS-PAGE分析,结果表明有一与预期分子量（20.6kDa）相应的诱导表达条带出现,其表达量约占全细胞蛋白的30%且以可溶形式存在。利用固定化金属离子（Ni^2＋）配体亲和层析柱纯化融合蛋白SSB,其纯度达到90%。通过凝胶层析和等离子共振技术对SSB的生物功能进行了系统研究分析。结果表明,SSB蛋白以四聚体形式与单链DNA分子结合,其亲和力常数（KD）为4.79×10^-7mol/L。

英文摘要：

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 （ Ni^2＋ ） 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.