Centre for Biotechnology, Anna University, Chennai, Tamil Nadu, India.
SRM Institute of Science and Technology, Chennai, Tamil Nadu, India.
PLoS One. 2020 Nov 30;15(11):e0242798. doi: 10.1371/journal.pone.0242798. eCollection 2020.
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.
真皮蛋白（DPT）是一种细胞外基质（ECM）蛋白，具有多样化的药物应用。它在细胞黏附/迁移、血管生成和 ECM 维持中发挥重要作用。该蛋白的重组生产将能够进一步探索其多方面的功能。在本研究中，旨在以经济有效的方式进行重组生产，我们在大肠杆菌（E.coli）中表达了 DPT 蛋白。将构建的重组载体（pRSETA-DPT）转化到大肠杆菌 GJ1158 表达系统中，并用 NaCl 诱导蛋白以包涵体的形式表达。包涵体在尿素中溶解，通过镍激活琼脂糖柱上的柱内复性程序进行蛋白复性。复性的组氨酸标记的 DPT 蛋白从柱上用咪唑洗脱，并通过分析技术确认其纯度。通过与标准 DPT 进行胶原纤维形成试验、伤口愈合试验和绒毛尿囊膜（CAM）血管生成试验，证实了该蛋白的生物活性。与对照组相比，纯化的 DPT 被发现可增强胶原纤维形成过程并促进人内皮细胞的迁移。在纯化的 DPT 处理的内皮细胞中，观察到约 73%的伤口闭合得到增强。纯化的 DPT 还可以在 CAM 模型中诱导血管生成。在现阶段，以适当的纯度和重现性扩大 DPT 的生产过程将具有广阔的商业前景。
