Functionally active rat S100A4 from a polymerase chain reaction-synthesized gene expressed in soluble form in Escherichia coli.
Functionally active rat S100A4 from a polymerase chain reaction-synthesized gene expressed in soluble form in Escherichia coli.
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功能活跃的大鼠 S100A4，来自 PCR 合成的基因，在大肠杆菌中以可溶形式表达
DOI：
10.3892/ol.2014.1870
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发表时间：
2014-04
期刊：
影响因子：
2.9
通讯作者：
Wang T
中科院分区：
文献类型：
--
作者：
Liu Z;Xu C;Zhang J;Chen Y;Liu X;Wu L;Zhang Z;Meng X;Liu H;Jiang Z;Wang T
S100A4蛋白参与钙离子依赖的细胞内活性调节，在肿瘤的侵袭、生长和转移中起重要作用。为了对大鼠S100A4进行功能表达和纯化后鉴定其生物学活性，通过重叠聚合酶链式反应优化并合成了S100A4基因片段。用噬菌体λ启动子将该基因插入原核表达载体pBV220，测序证实。构建表达载体pBV220-S100A4，转化大肠杆菌DH5α。温度诱导后，大鼠S100A4过表达，蛋白定位于裂解物上清液中，约占宿主内总蛋白的30-40%。用金属络合亲和层析法分离纯化该蛋白。获得了高纯度的S100A4蛋白(纯度为98%)，体外Western印迹分析表明，重组蛋白S100A4能与野生型S100A4抗体结合。通过Transwell迁移和侵袭实验检测重组蛋白的生物活性。制备了大鼠S100A4蛋白的多克隆抗体，用于兔免疫，与商品S100A4的免疫效果相当。因此，大鼠S100A4基因在大肠杆菌中得到了功能性表达，为进一步研究和应用S100A4蛋白奠定了基础。
S100A4 protein is associated with Ca2+-dependent regulation of intracellular activities and is significant in the invasion, growth and metastasis of cancer. In order to express rat S100A4 functionally and identify its biological activity following purification, an S100A4 gene fragment was optimized and fully synthesized via overlapping polymerase chain reaction. The gene was inserted into the prokaryotic expression vector, pBV220, with phage λ PRPL promoters following confirmation by DNA sequencing. The pBV220-S100A4 plasmid was constructed and transformed into Escherichia coli DH5α. Following temperature induction, rat S100A4 was overexpressed and the protein was observed to be located in the supernatant of the lysates, which was ~30–40% of the total protein within the host. The protein was isolated and purified by metal-chelate affinity chromatography. High purity protein (>98% purity) was obtained and in vitro western blot analysis identified that the recombinant S100A4 was able to bind to the antibody against wild-type S100A4. The bioactivity of the recombinant protein was detected via Transwell migration and invasion assays. The polyclonal antibody of rat S100A4 protein was prepared for rabbit immunization and exhibited similar efficacies when compared with commercial S100A4. Therefore, rat S100A4 was functionally expressed in E. coli; thus, the production of active recombinant S100A4 protein in E. coli may further aid with the investigation and application of S100A4.
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影响因子：
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作者：
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