[논문]Effective production of human growth factors in Escherichia coli by fusing with small protein 6HFh8

Kim, Young Su; Lee, Hye-Jeong; Han, Man-ho; Yoon, Nam-kyung; Kim, Yeu-chun; Ahn, Jungoh

doi:10.1186/s12934-020-01502-1

[해외논문] Effective production of human growth factors in Escherichia coli by fusing with small protein 6HFh8 원문보기

Microbial cell factories, v.20 no.1, 2021년, pp.9 -

Kim, Young Su (Department of Chemical and Biomolecular Engineering, KAIST, Daejeon, 34141 Republic of Korea) , Lee, Hye-Jeong (Biotechnology Process Engineering Center, KRIBB, Cheongju, 28116 Republic of Korea) , Han, Man-ho (Biotechnology Process Engineering Center, KRIBB, Cheongju, 28116 Republic of Korea) , Yoon, Nam-kyung (Biotechnology Process Engineering Center, KRIBB, Cheongju, 28116 Republic of Korea) , Kim, Yeu-chun (Department of Chemical and Biomolecular Engineering, KAIST, Daejeon, 34141 Republic of Korea) , Ahn, Jungoh (Biotechnology Process Engineering Center, KRIBB, Cheongju, 28116 Republic of Korea)

Abstract ▼ AI-Helper

BackgroundGrowth 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.ResultsWe 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.ConclusionsWe 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.

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