Cloning expression and characterization of dermatopontin for therapeutic applications

Abstract

Dermatopontin is a noncollagenous Extracellular Matrix Protein ECM with diversified applications in therapeutics Its expression has been reported in various mammalian tissues and is involved in various ECM and cellular activities DPT plays an important role in ECM structural maintenance extracellular matrix assembly cell migration cell adhesion and angiogenesis The recombinant production of this protein thus will help to further explore its functional activities and also enable its utilisation for its various applications In this study DPT has been produced in E coli aiming cost effective production The synthetic DPT gene was cloned into expression vector pRSETA and the constructed recombinant vector was transformed into expression host E coli GJ1158 The protein was expressed as inclusion bodies on induction with NaCl as His-tagged DPT protein The inclusion bodies were solubilised using urea and renatured by oncolumn refolding procedure in nickel chelated Sepharose columns The refolded Histidinetagged DPT protein was purified and eluted from column using imidazole and its purity was confirmed by analytical techniques The activity of refolded protein was confirmed by biological assays like fibril formation assay wound healing assay and angiogenesis assay using Chick Chorioallantoic membrane model Recombinant DPT rDPT exhibited activity comparable to that of standard DPT rDPT was found to promote collagen fibrillogenesis and could also enhance the migration of human endothelial cells About 73 enhanced wound closure was observed in purified DPT treated endothelial cells as compared to control newline