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Cloning, expression and purification of recombinant dermatopontin in Escherichia coli. 2020

Trikkur Madom Seetaraman Amritha, and Shubham Mahajan, and Kumar Subramaniam, and Yamini Chandramohan, and Anuradha Dhanasekaran
Centre for Biotechnology, Anna University, Chennai, Tamil Nadu, India.

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.

UI MeSH Term Description Entries
D011508 Chondroitin Sulfate Proteoglycans Proteoglycans consisting of proteins linked to one or more CHONDROITIN SULFATE-containing oligosaccharide chains. Proteochondroitin Sulfates,Chondroitin Sulfate Proteoglycan,Proteochondroitin Sulfate,Proteoglycan, Chondroitin Sulfate,Proteoglycans, Chondroitin Sulfate,Sulfate Proteoglycan, Chondroitin,Sulfate Proteoglycans, Chondroitin
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D002465 Cell Movement The movement of cells from one location to another. Distinguish from CYTOKINESIS which is the process of dividing the CYTOPLASM of a cell. Cell Migration,Locomotion, Cell,Migration, Cell,Motility, Cell,Movement, Cell,Cell Locomotion,Cell Motility,Cell Movements,Movements, Cell
D002479 Inclusion Bodies A generic term for any circumscribed mass of foreign (e.g., lead or viruses) or metabolically inactive materials (e.g., ceroid or MALLORY BODIES), within the cytoplasm or nucleus of a cell. Inclusion bodies are in cells infected with certain filtrable viruses, observed especially in nerve, epithelial, or endothelial cells. (Stedman, 25th ed) Cellular Inclusions,Cytoplasmic Inclusions,Bodies, Inclusion,Body, Inclusion,Cellular Inclusion,Cytoplasmic Inclusion,Inclusion Body,Inclusion, Cellular,Inclusion, Cytoplasmic,Inclusions, Cellular,Inclusions, Cytoplasmic
D003001 Cloning, Molecular The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells. Molecular Cloning
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D014945 Wound Healing Restoration of integrity to traumatized tissue. Healing, Wound,Healings, Wound,Wound Healings
D016326 Extracellular Matrix Proteins Macromolecular organic compounds that contain carbon, hydrogen, oxygen, nitrogen, and usually, sulfur. These macromolecules (proteins) form an intricate meshwork in which cells are embedded to construct tissues. Variations in the relative types of macromolecules and their organization determine the type of extracellular matrix, each adapted to the functional requirements of the tissue. The two main classes of macromolecules that form the extracellular matrix are: glycosaminoglycans, usually linked to proteins (proteoglycans), and fibrous proteins (e.g., COLLAGEN; ELASTIN; FIBRONECTINS; and LAMININ). Extracellular Matrix Protein,Matrix Protein, Extracellular,Matrix Proteins, Extracellular,Protein, Extracellular Matrix,Proteins, Extracellular Matrix
D017510 Protein Folding Processes involved in the formation of TERTIARY PROTEIN STRUCTURE. Protein Folding, Globular,Folding, Globular Protein,Folding, Protein,Foldings, Globular Protein,Foldings, Protein,Globular Protein Folding,Globular Protein Foldings,Protein Foldings,Protein Foldings, Globular

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