Department of Chemical and Biomolecular Engineering, KAIST, Daejeon, 34141, Republic of Korea.
Biotechnology Process Engineering Center, KRIBB, Cheongju, 28116, Republic of Korea.
Microb Cell Fact. 2021 Jan 7;20(1):9. doi: 10.1186/s12934-020-01502-1.
Growth factors (GFs) are signaling proteins that affect cellular processes such as growth, proliferation, and differentiation. GFs are used as cosmeceuticals, exerting anti-wrinkle, anti-aging, and whitening effects, and also as pharmaceuticals to treat wounds, growth failure, and oral mucositis. However, in mammalian and bacterial cells, low productivity and expression in inclusion bodies, respectively, of GFs does not satisfy the consumer demand. Here, we aimed to develop a bacterial expression system that produces high yields of soluble GFs that can be purified in their native forms.
We present Fh8, an 8-kDa peptide from Fasciola hepatica with an N-terminal hexa-histidine (6HFh8), as a fusion partner for enhanced human GF production in recombinant Escherichia coli. The fusion partner harboring a tobacco etch virus (TEV) protease cleavage site was fused to the N-terminus of 10 human GFs: acidic and basic fibroblast growth factors (aFGF and bFGF, respectively), epidermal growth factor (EGF), human growth hormone (hGH), insulin-like growth factor 1 (IGF-1), vascular endothelial growth factor 165 (VEGF165), keratinocyte growth factor 1 (KGF-1), placental growth factor (PGF), stem cell factor (SCF), and tissue inhibitor of metalloproteinase 1 (TIMP-1). The fusion proteins were expressed in E. coli under the control of T7 promoter at three temperatures (25 °C, 30 °C, and 37 °C). All individual fusion proteins, except for SCF and TIMP-1, were successfully overexpressed in cytoplasmic soluble form at more than one temperature. Further, the original aFGF, IGF-1, EGF, and VEGF165 proteins were cleaved from the fusion partner by TEV protease. Five-liter fed-batch fermentation approaches for the 6HFh8-aFGF (lacking disulfide bonds) and 6HFh8-VEGF165 (a cysteine-rich protein) were devised to obtain the target protein at concentrations of 9.7 g/l and 3.4 g/l, respectively. The two GFs were successfully highly purified (> 99% purity). Furthermore, they exerted similar cell proliferative effects as those of their commercial equivalents.
We demonstrated that 6HFh8-GF fusion proteins could be overexpressed on a g/l scale in the cytoplasm of E. coli, with the GFs subsequently highly purified and maintaining their biological activity. Hence, the small protein 6HFh8 can be used for efficient mass-production of various GFs.
生长因子（GFs）是一类信号蛋白，能够影响细胞的生长、增殖和分化等过程。GFs 可用作化妆品，具有抗皱、抗衰老和美白作用，也可用作药物来治疗伤口、生长不良和口腔粘膜炎。然而，在哺乳动物和细菌细胞中，GFs 的低产量和包涵体中的表达分别不能满足消费者的需求。在这里，我们旨在开发一种能够以可溶性形式产生大量可纯化的 GFs 的细菌表达系统。
我们提出了 Fasciola hepatica 的一种 8kDa 肽 Fh8，它带有一个 N 端六组氨酸（6HFh8），作为融合伴侣，用于增强重组大肠杆菌中人类 GFs 的产量。融合伴侣带有烟草蚀纹病毒（TEV）蛋白酶切割位点，融合到 10 个人类 GFs 的 N 端：酸性和成纤维细胞生长因子（aFGF 和 bFGF，分别）、表皮生长因子（EGF）、人生长激素（hGH）、胰岛素样生长因子 1（IGF-1）、血管内皮生长因子 165（VEGF165）、角质细胞生长因子 1（KGF-1）、胎盘生长因子（PGF）、干细胞因子（SCF）和基质金属蛋白酶 1 抑制剂（TIMP-1）。融合蛋白在 T7 启动子的控制下，在 25°C、30°C 和 37°C 三种温度下在大肠杆菌中表达。除了 SCF 和 TIMP-1 之外，所有的融合蛋白都在至少一种温度下以细胞质可溶性形式成功过量表达。此外，原始的 aFGF、IGF-1、EGF 和 VEGF165 蛋白通过 TEV 蛋白酶从融合伴侣上切割下来。为了获得浓度分别为 9.7g/L 和 3.4g/L 的目标蛋白，设计了用于 6HFh8-aFGF（缺乏二硫键）和 6HFh8-VEGF165（富含半胱氨酸的蛋白质）的 5 升补料分批发酵方法。两种 GFs 均成功高度纯化（>99%纯度）。此外，它们表现出与商业等价物相似的细胞增殖作用。
我们证明了 6HFh8-GF 融合蛋白可以在大肠杆菌的细胞质中以克/升的规模过量表达，GFs 随后得到高度纯化并保持其生物活性。因此，小蛋白 6HFh8 可用于各种 GFs 的高效大规模生产。
