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Applied Microbiology and Biotechnology

, Volume 93, Issue 2, pp 613–621 | Cite as

Large-scale expression, purification, and glucose uptake activity of recombinant human FGF21 in Escherichia coli

  • Minjing Zhang
  • Xuebin Jiang
  • Zhijian Su
  • Jiancong Lin
  • Qi Xiang
  • Zhan Yang
  • Yadong Huang
  • Xiaokun Li
Biotechnologically Relevant Enzymes and Proteins

Abstract

As a novel important regulator of glucose and lipid metabolism homeostasis, human fibroblast growth factor 21 (hFGF21) has become a potential drug candidate for the treatment of metabolic diseases including obesity, and type 2 diabetes, as well as non-alcoholic fatty liver disease. To improve the production of recombinant hFGF21 to meet the increasing demand in clinical applications, an artificial gene encoding its mature peptide sequence was constructed, cloned into vector pET-3c and then expressed in Escherichia coli Origami B (DE3). Under optimal conditions in a 50-L fermentor, the average bacterial yield and the soluble expression level of recombinant hFGF21 of six batches attained 1750 ± 185 g and 32 ± 1.5%, respectively. The target protein was purified by the combination of nickel-nitrilotriacetic acid affinity chromatography and Sephadex S-100 resin. 5% (w/v) trehalose solution was able to prevent rhFGF21 from degradation effectively. The purity of rhFGF21 was higher than 97%, and the yield was 213 ± 17 mg/L. The preliminary biochemical characterization of rhFGF21 was confirmed using Western blot and peptide map finger analysis. Based on the glucose oxidase–peroxidase assay, the EC50 of glucose uptake activity of the purified rhFGF21 was 22.1 nM.

Keywords

Human fibroblast growth factor 21 Expression Purification Glucose uptake activity 

Notes

Acknowledgments

This work was supported by grants of the National Natural Science Foundation of China (No.31000663), National Key New Drug Foundation of China (No.2011ZX09102-004-03 and 2009ZX09103-749), the Science Foundation of Wenzhou of China (No.Y20090279), the Science Foundation of Guangdong of China (No. 2009B080701090 and 2007B030700004), and the Program for New Century Excellent Talents in University (NCET-08-0611).

References

  1. Coskun T, Bina HA, Schneider MA, Dunbar JD, Hu CC, Chen Y, Moller DE, Kharitonenkov A (2008) FGF21 corrects obesity in mice. Endocrinology 149:6018–6027CrossRefGoogle Scholar
  2. Goetz R, Beenken A, Ibrahimi OA, Kalinina J, Olsen SK, Eliseenkova AV, Xu C, Neubert TA, Zhang F, Linhardt RJ, Yu X, White KE, Inagaki T, Kliewer SA, Yamamoto M, Kurosu H, Ogawa Y, Kuro-o M, Lanske B, Razzaque MS, Mohammadi M (2007) Molecular insights into the klotho-dependent, endocrine mode of action of fibroblast growth factor 19 subfamily members. Mol Cell Biol 27:3417–3428CrossRefGoogle Scholar
  3. Kharitonenkov A, Shanafelt AB (2009) FGF21: a novel prospect for the treatment of metabolic diseases. Curr Opin Investig Drugs 10:359–364Google Scholar
  4. 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 Invest 115:1627–1635CrossRefGoogle Scholar
  5. Kharitonenkov A, Wroblewski VJ, Koester A, Chen YF, Clutinger CK, Tigno XT, Hansen BC, Shanafelt AB, Etgen GJ (2007) The metabolic state of diabetic monkeys is regulated by fibroblast growth factor-21. Endocrinology 148:774–781CrossRefGoogle Scholar
  6. Kharitonenkov A, Dunbar JD, Bina HA, Bright S, Moyers JS, Zhang C, Ding L, Micanovic R, Mehrbod SF, Knierman MD, Hale JE, Coskun T, Shanafelt AB (2008) FGF21/FGF21, receptor interaction and activation is determined by beta Klotho. J Cell Physiol 215:1–7CrossRefGoogle Scholar
  7. Luli GW, Strohl WR (1990) Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations. Appl Environ Microbiol 56(4):1004–1011Google Scholar
  8. Micanovic R, Raches DW, Dunbar JD, Driver DA, Bina HA, Dickinson CD, Kharitonenkov A (2009) Different roles of N- and C-termini in the functional activity of FGF21. J Cell Physiol 219:227–234CrossRefGoogle Scholar
  9. Nishimura T, Nakatake Y, Konishi M, Itoh N (2000) Identification of a novel FGF, FGF-21, preferentially expressed in the liver. Biochim Biophys Acta 492:203–206Google Scholar
  10. Prinz WA, Aslund F, Holmgren A, Beckwith J (1997) The role of the thioredoxin and glutaredoxin pathways in reducing protein disulfide bonds in the Escherichia coli cytoplasm. J Biol Chem 272:15661–15667CrossRefGoogle Scholar
  11. 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 Biotechnology 10:14CrossRefGoogle Scholar
  12. Wente W, Efanov AM, Brenner M, Kharitonenkov A, Köster A, Sandusky GE, Sewing S, Treinies I, Zitzer H, Gromada J (2006) Fibroblast growth factor-21 improves pancreatic beta-cell function and survival byactivation of extracellular signal-regulated kinase 1/2 and Akt signaling pathways. Diabetes 55:2470–2478CrossRefGoogle Scholar
  13. 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, Véniant MM (2009) Fibroblast growth factor 21 reverses hepatic steatosis, increases energy expenditure, and improves insulin sensitivity in diet-induced obese mice. Diabetes 58:250–259CrossRefGoogle Scholar
  14. 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, Véniant M, Xu J (2009) FGF21 N- and C-termini play different roles in receptor interaction and activation. FEBS Lett 583:19–24CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Minjing Zhang
    • 1
    • 2
    • 4
  • Xuebin Jiang
    • 3
  • Zhijian Su
    • 1
    • 2
    • 4
  • Jiancong Lin
    • 3
  • Qi Xiang
    • 2
  • Zhan Yang
    • 2
  • Yadong Huang
    • 1
    • 2
  • Xiaokun Li
    • 4
  1. 1.Institute of Life and Health Engineering, and National Engineering Research Center of Genetic MedicineJinan UniversityGuangzhouPeople’s Republic of China
  2. 2.Guangdong Provincial Key Laboratory of Bioengineering MedicineJinan UniversityGuangzhouPeople’s Republic of China
  3. 3.School of Life SciencesGuangzhou UniversityGuangzhouPeople’s Republic of China
  4. 4.Wenzhou Medical CollegeWenzhouPeople’s Republic of China

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