Cloning, expression and purification of recombinant dermatopontin in Escherichia coli

Abstract

Dermatopontin (DPT) is an extracellular matrix (ECM) protein with diversified pharmaceutical applications. It plays important role in cell adhesion/migration, angiogenesis and ECM maintenance. The recombinant production of this protein will enable further exploration of its multifaceted functions. In this study, DPT protein has been expressed in Escherichia coli (E.coli) aiming at cost effective recombinant production. The E.coli GJ1158 expression system was transformed with constructed recombinant vector (pRSETA-DPT) and protein was expressed as inclusion bodies on induction with NaCl. The inclusion bodies were solubilised in urea and renaturation of protein was done by on-column refolding procedure in Nickel activated Sepharose column. The refolded Histidine-tagged DPT protein was purified and eluted from column using imidazole and its purity was confirmed by analytical techniques. The biological activity of the protein was confirmed by collagen fibril assay, wound healing assay and Chorioallantoic Membrane (CAM) angiogenesis assay on comparison with standard DPT. The purified DPT was found to enhance the collagen fibrillogenesis process and improved the migration of human endothelial cells. About 73% enhanced wound closure was observed in purified DPT treated endothelial cells as compared to control. The purified DPT also could induce neovascularisation in the CAM model. At this stage, scaling up the production process for DPT with appropriate purity and reproducibility will have a promising commercial edge.

Generation of a recombinant version of a biologically active cell-permeant human HAND2 transcription factor from E. coli

“…The endothelial cell migration and angiogenic potential of this purified fusion protein was comparable to the VEGF, a crucial inducer of angiogenesis. Similar observations of neovascularization were reported, induced by E. coli- derived recombinant asparaginyl-tRNA synthetase 35 and dermatopontin 36 proteins. It is reported that HAND2 regulates angiogenesis through the Notch signaling pathway 59 .…”

“…In order to study the angiogenic potential of HAND2 protein, we performed a chicken CAM assay. The chick embryo model is a widely used ex vivo model to study the angiogenic/anti-angiogenic potential of various biological molecules including recombinant proteins 35 , 36 . For the slow diffusion of recombinant protein, we soaked the filter paper discs in the protein solution and gently placed them on the CAMs.…”

“…The confluent monolayers were scratched with a sterile pipette tip (0–20 μL), and the spent media was discarded. The scratched monolayers were rinsed with sterile PBS and then treated with rhHTN-HAND2 protein or an equivalent volume of vehicle control (in endothelial cell expansion media) or VEGF 35 , 36 for 12 h. Images were captured at 20 × magnification using an inverted brightfield microscope (ZOE Fluorescent Cell Imager, Bio-Rad) at different time intervals. The images were exported in .tiff file format for the quantification of the scratched area using ImageJ (1.48v) software.…”

“…The CAM assay was performed as described previously 35 , 36 with minor modifications. The 3–4 days old embryonated chicken eggs were directly obtained from the local chicken egg hatching unit.…”