Two-hundred-liter scale fermentation, purification of recombinant human fibroblast growth factor-21, and its anti-diabetic effects on ob/ob mice
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Fibroblast growth factor-21 (FGF-21) is a potential cytokine for type II diabetes mellitus. This study aimed to optimize recombinant human FGF-21 (rhFGF-21) production in Escherichia coli BL21 (DE3) employing high cell density fermentation at a 200-L scale and pilot-scale purification. FGF-21 was eventually expressed in E. coli BL21 (DE3) using human FGF-21 synthetic DNA sequence via the introduction of vector pET-3c; the product is used as seed strain during the fermentation of rhFGF-21. Fermentation of rhFGF-21 was performed in a 30-L and 200-L fermenters. rhFGF-21 was primarily expressed in the form of inclusion bodies after IPTG induction. At the 200-L scale, the bacterial production and expression levels of rhFGF-21 were 38.8 ± 0.6 g/L and 30.9 ± 0.7%, respectively. Additionally, the high purification (98%) of rhFGF-21 was tested with HPLC analysis and reducing & non-reducing SDS-PAGE analysis. The final yield of purified rhFGF-21 was 71.1 ± 13.9 mg/L. The activity of rhFGF-21 stock solution reached at 68.67 ± 8.74 IU/mg. Blood glucose controlling and insulin sensitization were improved with treatment of rhFGF-21 in type II diabetic ob/ob mice. Our results showed that the relatively stable and time-saving pilot-scale production process was successfully established, providing an efficient and cost-effective strategy for large-scale and industrial production of rhFGF-21.
KeywordsFGF-21 Expression Purification Glucose-lowering
This research was funded by a grant from the ministry of science and technology of China (No. 2011ZX09102-004-03), the National Natural Science Foundation of China (No. 81601695), the Natural Science Foundation of Zhejiang Province (No. LY17H150002), and the Wenzhou Science and Technology Agency (No. ZS2017016).
Compliance with ethical standards
All animal experiments were handled in accordance with IACUC guidelines of Wenzhou Medical University (Zhejiang, China), complying with NIH guidelines for the care and use of laboratory animals. This article does not contain any studies with human participants. All authors confirm that ethical principles have been followed in the experiments.
Conflicts of interest
The authors declare that they have no conflict of interest.
- Bookout AL, de Groot MH, Owen BM, Lee S, Gautron L, Lawrence HL, Ding X, Elmquist JK, Takahashi JS, Mangelsdorf DJ, Kliewer SA (2013) FGF21 regulates metabolism and circadian behavior by acting on the nervous system. Nat Med 19(9):1147–1152. https://doi.org/10.1038/nm.3249 CrossRefPubMedPubMedCentralGoogle Scholar
- Domouzoglou EM, Naka KK, Vlahos AP, Papafaklis MI, Michalis LK, Tsatsoulis A, Maratos-Flier E (2015) Fibroblast growth factors in cardiovascular disease: the emerging role of FGF21. Am J Physiol Heart Circ Physiol 309(6):H1029–H1038. https://doi.org/10.1152/ajpheart.00527.2015 CrossRefPubMedPubMedCentralGoogle Scholar
- Goto T, Hirata M, Aoki Y, Iwase M, Takahashi H, Kim M, Li Y, Jheng HF, Nomura W, Takahashi N, Kim CS, Yu R, Seno S, Matsuda H, Aizawa-Abe M, Ebihara K, Itoh N, Kawada T (2017) The hepatokine FGF21 is crucial for peroxisome proliferator-activated receptor-alpha agonist-induced amelioration of metabolic disorders in obese mice. J Biol Chem 292(22):9175–9190. https://doi.org/10.1074/jbc.M116.767590 CrossRefPubMedPubMedCentralGoogle Scholar
- Holland WL, Adams AC, Brozinick JT, Bui HH, Miyauchi Y, Kusminski CM, Bauer SM, Wade M, Singhal E, Cheng CC, Volk K, Kuo MS, Gordillo R, Kharitonenkov A, Scherer PE (2013) An FGF21-adiponectin-ceramide axis controls energy expenditure and insulin action in mice. Cell Metab 17(5):790–797. https://doi.org/10.1016/j.cmet.2013.03.019 CrossRefPubMedPubMedCentralGoogle Scholar
- Kharitonenkov A, Shiyanova TL, Koester A, Ford AM, Micanovic R, Galbreath EJ, Sandusky GE, Hammond LJ, Moyers JS, Owens RA, Gromada J, Brozinick JT, Hawkins ED, Wroblewski VJ, Li DS, Mehrbod F, Jaskunas SR, Shanafelt AB (2005) FGF-21 as a novel metabolic regulator. J Clin Investig 115(6):1627–1635. https://doi.org/10.1172/JCI23606 CrossRefPubMedGoogle Scholar
- Kim HW, Lee JE, Cha JJ, Hyun YY, Kim JE, Lee MH, Song HK, Nam DH, Han JY, Han SY, Han KH, Kang YS, Cha DR (2013) Fibroblast growth factor 21 improves insulin resistance and ameliorates renal injury in db/db mice. Endocrinology 154(9):3366–3376. https://doi.org/10.1210/en.2012-2276 CrossRefPubMedGoogle Scholar
- Lee JH, Lee JE, Kang KJ, Jang YJ (2017) Functional efficacy of human recombinant FGF-2s tagged with (His)6 and (His-Asn)6 at the N- and C-termini in human gingival fibroblast and periodontal ligament-derived cells. Protein Expr Purif 135:37–44. https://doi.org/10.1016/j.pep.2017.05.001 CrossRefPubMedGoogle Scholar
- Vemula S, Thunuguntla R, Dedaniya A, Kokkiligadda S, Palle C, Ronda SR (2015) Improved production and characterization of recombinant human granulocyte colony stimulating factor from E. coli under optimized downstream processes. Protein Expr Purif 108:62–72. https://doi.org/10.1016/j.pep.2015.01.010 CrossRefPubMedGoogle Scholar
- Vu TT, Jeong B, Krupa M, Kwon U, Song JA, Do BH, Nguyen MT, Seo T, Nguyen AN, Joo CH, Choe H (2016) Soluble prokaryotic expression and purification of human interferon alpha-2b using a maltose-binding protein tag. J Mol Microbiol Biotechnol 26(6):359–368. https://doi.org/10.1159/000446962 CrossRefPubMedGoogle Scholar
- Wang S, Lin H, Zhao T, Huang S, Fernig DG, Xu N, Wu F, Zhou M, Jiang C, Tian H (2017) Expression and purification of an FGF9 fusion protein in E. coli, and the effects of the FGF9 subfamily on human hepatocellular carcinoma cell proliferation and migration. Appl Microbiol Biotechnol 101(21):7823–7835. https://doi.org/10.1007/s00253-017-8468-1 CrossRefPubMedGoogle Scholar
- Wente W, Efanov AM, Brenner M, Kharitonenkov A, Koster A, Sandusky GE, Sewing S, Treinies I, Zitzer H, Gromada J (2006) Fibroblast growth factor-21 improves pancreatic beta-cell function and survival by activation of extracellular signal-regulated kinase 1/2 and Akt signaling pathways. Diabetes 55(9):2470–2478. https://doi.org/10.2337/db05-1435 CrossRefPubMedGoogle Scholar
- Xu J, Lloyd DJ, Hale C, Stanislaus S, Chen M, Sivits G, Vonderfecht S, Hecht R, Li YS, Lindberg RA, Chen JL, Jung DY, Zhang Z, Ko HJ, Kim JK, Veniant MM (2009) Fibroblast growth factor 21 reverses hepatic steatosis, increases energy expenditure, and improves insulin sensitivity in diet-induced obese mice. Diabetes 58(1):250–259. https://doi.org/10.2337/db08-0392 CrossRefPubMedPubMedCentralGoogle Scholar
- Yang X, Hui Q, Yu B, Huang Z, Zhou P, Wang P, Wang Z, Pang S, Li J, Wang H, Lin L, Li X, Wang X (2018) Design and evaluation of lyophilized fibroblast growth factor 21 and its protection against ischemia cerebral injury. Bioconjug Chem 29(2):287–295. https://doi.org/10.1021/acs.bioconjchem.7b00588 CrossRefPubMedGoogle Scholar
- Yie J, Hecht R, Patel J, Stevens J, Wang W, Hawkins N, Steavenson S, Smith S, Winters D, Fisher S, Cai L, Belouski E, Chen C, Michaels ML, Li YS, Lindberg R, Wang M, Veniant M, Xu J (2009) FGF21 N- and C-termini play different roles in receptor interaction and activation. FEBS Lett 583(1):19–24. https://doi.org/10.1016/j.febslet.2008.11.023 CrossRefPubMedGoogle Scholar