title: Functionally active rat S100A4 from a polymerase chain reaction-synthesized gene expressed in soluble form inEscherichia coli.
abstract: 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 PRPLpromoters following confirmation by DNA sequencing. The pBV220-S100A4 plasmid was constructed and transformed intoEscherichia coliDH5α. 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 andin vitrowestern 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 inE. coli; thus, the production of active recombinant S100A4 protein inE. colimay further aid with the investigation and application of S100A4.
authors.affiliation: Institute of Health and Environmental Medicine, Heping, Tianjin 300050, P.R. China ; Department of Physiology and Pathophysiology, Logistics College of Chinese People's Armed Police Force, Hedong, Tianjin 300162, P.R. China.
authors.forename: Ziquan
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Institute of Health and Environmental Medicine, Heping, Tianjin 300050, P.R. China.
authors.forename: Chuanxiang
authors.initials: C
authors.lastname: Xu
authors.affiliation: Institute of Health and Environmental Medicine, Heping, Tianjin 300050, P.R. China ; Tianjin University of Sport, Nankai, Tianjin 300381, P.R. China.
authors.forename: Jianwei
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Institute of Health and Environmental Medicine, Heping, Tianjin 300050, P.R. China ; Tianjin University of Sport, Nankai, Tianjin 300381, P.R. China.
authors.forename: Yunyun
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Institute of Health and Environmental Medicine, Heping, Tianjin 300050, P.R. China.
authors.forename: Xiaohua
authors.initials: X
authors.lastname: Liu
authors.affiliation: Institute of Health and Environmental Medicine, Heping, Tianjin 300050, P.R. China.
authors.forename: Lei
authors.initials: L
authors.lastname: Wu
authors.affiliation: Institute of Health and Environmental Medicine, Heping, Tianjin 300050, P.R. China.
authors.forename: Zhiqing
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Institute of Health and Environmental Medicine, Heping, Tianjin 300050, P.R. China ; Department of Physiology and Pathophysiology, Logistics College of Chinese People's Armed Police Force, Hedong, Tianjin 300162, P.R. China.
authors.forename: Xiangyan
authors.initials: X
authors.lastname: Meng
authors.affiliation: Institute of Health and Environmental Medicine, Heping, Tianjin 300050, P.R. China.
authors.forename: Hongtao
authors.initials: H
authors.lastname: Liu
authors.affiliation: Institute of Zoology, Chinese Academy of Sciences, Chaoyang, Beijing 100101, P.R. China.
authors.forename: Zifeng
authors.initials: Z
authors.lastname: Jiang
authors.affiliation: Institute of Health and Environmental Medicine, Heping, Tianjin 300050, P.R. China.
authors.forename: Tianhui
authors.initials: T
authors.lastname: Wang
keywords: functional expression; gene recombination; rat S100A4