Laboratory of Structural Biology and Protein Engineering, Carlos Chagas Institute, FIOCRUZ Paraná, Curitiba, PR, Brazil.
Cellular and Molecular Biology Graduate Program, Federal University of Paraná, Curitiba, PR, Brazil.
Sci Rep. 2022 Mar 23;12(1):5034. doi: 10.1038/s41598-022-09060-9.
Epidermal growth factors (EGF) play a wide range of roles in embryogenesis, skin development, immune response homeostasis. They are involved in several pathologies as well, including several cancer types, psoriasis, chronic pain and chronic kidney disease. All members share the structural EGF domain, which is responsible for receptor interaction, thereby initiating transduction of signals. EGF growth factors have intense use in fundamental research and high potential for biotechnological applications. However, due to their structural organization with three disulfide bonds, recombinant production of these factors in prokaryotic systems is not straightforward. A significant fraction usually forms inclusion bodies. For the fraction remaining soluble, misfolding and incomplete disulfide bond formation may affect the amount of active factor in solution, which can compromise experimental conclusions and biotechnological applications. In this work, we describe a reliable procedure to produce seven human growth factors of the EGF family in Escherichia coli. Biophysical and stability analyses using limited proteolysis, light scattering, circular dichroism and nanoDSF show that the recombinant factors present folded and stable conformation. Cell proliferation and scratch healing assays confirmed that the recombinant factors are highly active at concentrations as low as 5 ng/ml.
表皮生长因子（EGF）在胚胎发生、皮肤发育、免疫反应稳态中发挥广泛作用。它们也参与多种病理学，包括几种癌症类型、银屑病、慢性疼痛和慢性肾病。所有成员都具有结构 EGF 结构域，该结构域负责与受体相互作用，从而启动信号转导。EGF 生长因子在基础研究中有广泛应用，在生物技术应用方面有很大潜力。然而，由于其具有三个二硫键的结构组织，这些因子在原核系统中的重组生产并不简单。很大一部分通常形成包含体。对于剩余可溶性的部分，错误折叠和不完全的二硫键形成可能会影响溶液中活性因子的数量，这可能会影响实验结论和生物技术应用。在这项工作中，我们描述了一种在大肠杆菌中生产七种 EGF 家族人类生长因子的可靠方法。使用有限蛋白酶解、光散射、圆二色性和纳米 DSF 进行的生物物理和稳定性分析表明，重组因子呈现折叠和稳定的构象。细胞增殖和划痕愈合试验证实，重组因子在低至 5ng/ml 的浓度下仍具有高度活性。
