Expression And Purification Of S100A4 Fusion Protein In E.coli Bl21 In Sika Deer Antler Stem Cells

As the only mammal organ that can completely regenerate after lost, antler is an ideal animal model for the study of mammalian organ regeneration and wound healing. Studies have shown that the generation and regeneration of antler are both dependent upon the stem cells. Antler stem cells exist in the periosteum where antler generates, in other words, the Antler Periosteum (AP). S100A4, which works as the specific molecule during the AP cells expression, is presumed as the key regulatory molecule in antler generation. Purified S100A4 with high quality is needed in order to further elaborate the regulatory mechanisms of antler generation at the molecular level.â… n this study, based on the S100A4 gene sequence that had been produced by reverse transcription from sika deer's AP cells, upstream and downstream primers containing EcoRâ… and BamHâ… restriction sites were designed and desired gene fragments were amplified. The fragment is 330bp in size and the expression vector thatâ… used is PGEX-6P-1. The amplified S100A4 gene fragment and PGEX-6P-1 were cut by EcoRâ… and BamHâ… respectively, then the fragment and vector were recycled and connected using T4 ligase. By transferring the ligation product into Top10F' competent cells,â… sifted positive colonies out on LB plate with ampicillin resistance. After being confirmed by PCR and Double Digestion, the positive strains were then sequenced. The recombinant plasmid which had been correctly connected was extracted and was transfected into BL21 (DE3) pLys S competent cells to screen positive monoclonal strains. The resulting strains were induced by isopropyl thiogalactoside (â… PTG) for expression. Polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot confirmed that S100A4-GST fusion protein was successfully expressed. The E.coli was shattered using ultrasonication, then centrifuged to remove precipitate, and the result of SDS-PAGE electrophoresis of supernatant indicated that the fusion protein was in soluble expression fraction. Fusion protein was largely induced in quantity for expression and purified with GST column.We successfully expressed the deer antler stem cell S100A4-GST fusion protein in vitro and had it purified, which provided the condition for further study of the role S100A4 protein playing in the mechanism of antler generation.