Abstract
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.
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References
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
Cao F, Liu X, Cao X, Wang S, Fu K, Zhao Y, Shen F, Liu J (2017) Fibroblast growth factor 21 plays an inhibitory role in vascular calcification in vitro through OPG/RANKL system. Biochem Biophys Res Commun 491(3):578–586. https://doi.org/10.1016/j.bbrc.2017.07.160
Chiavaroli A, Recinella L, Ferrante C, Martinotti S, Vacca M, Brunetti L, Orlando G, Leone S (2017) Effects of central fibroblast growth factor 21 and irisin in anxiety-like behavior. J Biol Regul Homeost Agents 31(3):797–802
Coskun T, Bina HA, Schneider MA, Dunbar JD, Hu CC, Chen Y, Moller DE, Kharitonenkov A (2008) Fibroblast growth factor 21 corrects obesity in mice. Endocrinology 149(12):6018–6027. https://doi.org/10.1210/en.2008-0816
Dagar VK, Adivitiya, Khasa YP (2017) High-level expression and efficient refolding of therapeutically important recombinant human Interleukin-3 (hIL-3) in E. coli. Protein Expr Purif 131:51–59. https://doi.org/10.1016/j.pep.2016.11.005
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
Fukumoto S (2008) Actions and mode of actions of FGF19 subfamily members. Endocr J 55(1):23–31. https://doi.org/10.1507/endocrj.KR07E-002
Gaich G, Chien JY, Fu H, Glass LC, Deeg MA, Holland WL, Kharitonenkov A, Bumol T, Schilske HK, Moller DE (2013) The effects of LY2405319, an FGF21 analog, in obese human subjects with type 2 diabetes. Cell Metab 18(3):333–340. https://doi.org/10.1016/j.cmet.2013.08.005
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
He Y, Li Y, Wei Z, Zhang X, Gao J, Wang X, Li X, Wu W, Li W, Yi X, Zeng Y, Liu C (2018) Pharmacokinetics, tissue distribution, and excretion of FGF-21 following subcutaneous administration in rats. Drug Test Anal 10:1061–1069. https://doi.org/10.1002/dta.2365
Hojman P, Pedersen M, Nielsen AR, Krogh-Madsen R, Yfanti C, Akerstrom T, Nielsen S, Pedersen BK (2009) Fibroblast growth factor-21 is induced in human skeletal muscles by hyperinsulinemia. Diabetes 58(12):2797–2801. https://doi.org/10.2337/db09-0713
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
Hondares E, Iglesias R, Giralt A, Gonzalez FJ, Giralt M, Mampel T, Villarroya F (2011) Thermogenic activation induces FGF21 expression and release in brown adipose tissue. J Biol Chem 286(15):12983–12990. https://doi.org/10.1074/jbc.M110.215889
Huang Z, Xu A, Cheung BMY (2017) The potential role of fibroblast growth factor 21 in lipid metabolism and hypertension. Curr Hypertens Rep 19(4):28. https://doi.org/10.1007/s11906-017-0730-5
Jin L, Lin Z, Xu A (2016) Fibroblast growth factor 21 protects against atherosclerosis via fine-tuning the multiorgan crosstalk. Diabetes Metab J 40(1):22–31. https://doi.org/10.4093/dmj.2016.40.1.22
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
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
Kwok KHM, Lam KSL (2017) Fibroblast growth factor 21 mimetics for treating atherosclerosis. Endocrinol Metab (Seoul) 32(2):145–151. https://doi.org/10.3803/EnM.2017.32.2.145
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
Nishimura T, Nakatake Y, Konishi M, Itoh N (2000) Identification of a novel FGF, FGF-21, preferentially expressed in the liver. Biochim Biophys Acta 1492(1):203–206
Pan ZC, Wang SP, Ou TT, Liu H, Ma JW, Wang WX, Fang WY, Qu XK, Zhang M (2017) A study on the expression of FGF-21 and NF-kappaB pathway in the tissues of atherosclerotic mice. Eur Rev Med Pharmacol Sci 21(3 Suppl):102–107
Ribas F, Villarroya J, Hondares E, Giralt M, Villarroya F (2014) FGF21 expression and release in muscle cells: involvement of MyoD and regulation by mitochondria-driven signalling. Biochem J 463(2):191–199. https://doi.org/10.1042/BJ20140403
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
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
Wang H, Xiao Y, Fu L, Zhao H, Zhang Y, Wan X, Qin Y, Huang Y, Gao H, Li X (2010) High-level expression and purification of soluble recombinant FGF21 protein by SUMO fusion in Escherichia coli. BMC Biotechnol 10:14. https://doi.org/10.1186/1472-6750-10-14
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
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
Xie T, Leung PS (2017) Fibroblast growth factor 21: a regulator of metabolic disease and health span. Am J Physiol Endocrinol Metab 313(3):E292–E302. https://doi.org/10.1152/ajpendo.00101.2017
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
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
Ye X, Qi J, Yu D, Li S, Wu Q, Wu Y, Ren G, Han J, Li D (2016) Pilot-scale production and characterization of PEGylated human FGF-21 analog. J Biotechnol 228:8–17. https://doi.org/10.1016/j.jbiotec.2016.04.038
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
Zhang M, Jiang X, Su Z, Lin J, Xiang Q, Yang Z, Huang Y, Li X (2012) Large-scale expression, purification, and glucose uptake activity of recombinant human FGF21 in Escherichia coli. Appl Microbiol Biotechnol 93(2):613–621. https://doi.org/10.1007/s00253-011-3427-8
Acknowledgements
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).
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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.
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Hui, Q., Huang, Z., Pang, S. et al. Two-hundred-liter scale fermentation, purification of recombinant human fibroblast growth factor-21, and its anti-diabetic effects on ob/ob mice. Appl Microbiol Biotechnol 103, 719–730 (2019). https://doi.org/10.1007/s00253-018-9470-y
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DOI: https://doi.org/10.1007/s00253-018-9470-y