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.coli K-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（20.6 kD）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（Ni2+）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4.79×10-7 M。
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.
乔惠丽,陈媛媛,文祯中,毕利军,阚云超. SSB蛋白的高效表达及其与ssDNA的相互作用[J]. 中国生物工程杂志, 2007, 27(4): 12-17.
. Cloning, High Expression of Single-Stranded DNA-Binding Protein and Its Interaction with ssDNA. China Biotechnology, 2007, 27(4): 12-17.
https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2007/V27/I4/12
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