Functionally active rat S100A4 from a polymerase chain reaction-synthesized gene expressed in soluble form in Escherichia coli

April 2014
Oncology Letters;2014, Vol. 7 Issue 4, p1179
Academic Journal
S100A4 protein is associated with Ca2+-dependent regulation of intracellular activities and is significant in 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 overexpressed and the protein was observed to be located the supernatant of the lysates, which was ~30-40% of the 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 the antibody against wild-type S100A4. The bioactivity of 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. thus, the production of active recombinant S100A4 protein E. coli may further aid with the investigation and application of S100A4.


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