Purification of recombinant human fibroblast growth factor 13 in E. coli and its molecular mechanism of mitogenesis
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Fibroblast growth factor (FGF) 13, a member of the FGF11 subfamily, is a kind of intracrine protein similar to other family members including FGF11, FGF12, and FGF14. Unlike classical FGF, FGF13 exerts its bioactivities independent of fibroblast growth factor receptors (FGFRs). However, the effect of exogenous administration of FGF13 still remains further investigated. In the present study, we established an Escherichia coli expression system for the large-scale production of FGF13 and then obtained two isoform proteins including recombinant human FGF13A (rhFGF13A) and rhFGF13B with a purity greater than 90% by column chromatography, respectively. Otherwise, soluble analysis indicated that both rhFGF13A and rhFGF13B expressed in E. coli BL21 (DE3) pLysS were soluble. Furthermore, cellular-based experiments demonstrated that rhFGF13A, rather than rhFGF13B, could promote the proliferation of NIH3T3 cells in the presence of heparin. Mechanistically, the mitogenic effect of FGF13 was mediated by activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), but not p38. Moreover, blockage of FGFRs also significantly attenuated the mitogenic effects of rhFGF13A, implying that FGFRs are still related to FGF13. Thus, our research shows that exogenous FGF13 can act as secreted FGF to participate in cell signal transmission and heparin is still required as an ancillary cofactor for the mitogenic effects of FGF13, which may help people to discover more potential functions of FGF13 in cell life activities.
KeywordsrhFGF13 Protein purification Mitogen activity Heparin NIH3T3 cell Cell signaling pathway
This work was supported by the Opening Project of Zhejiang Provincial TOP Key Discipline of Pharmaceutical Sciences (No.201720).
Compliance with ethical standards
This article does not contain any studies with animals or human participants. All authors confirm that ethical principles have been followed in the experiments.
Conflict of interest
The authors declare that they have no conflict of interest.
- Cheng J, Fang Z, Yang H, Li Y, Tian H, Gong W, Chen T, Liu M, Li X, Jiang C (2017) High-yield of biologically active recombinant human fibroblast growth factor-16 in E. coli and its mechanism of proliferation in NCL-H460 cells. Prep Biochem Biotechnol 47(7):720–729. https://doi.org/10.1080/10826068.2017.1315599 CrossRefGoogle Scholar
- DeStefano GM, Fantauzzo KA, Petukhova L, Kurban M, Tadin-Strapps M, Levy B, Warburton D, Cirulli ET, Han Y, Sun X, Shen Y, Shirazi M, Jobanputra V, Cepeda-Valdes R, Cesar Salas-Alanis J, Christiano AM (2013) Position effect on FGF13 associated with X-linked congenital generalized hypertrichosis. Proc Natl Acad Sci U S A 110(19):7790–7795. https://doi.org/10.1073/pnas.1216412110 CrossRefGoogle Scholar
- Gecz J, Baker E, Donnelly A, Ming JE, McDonald-McGinn DM, Spinner NB, Zackai EH, Sutherland GR, Mulley JC (1999) Fibroblast growth factor homologous factor 2 (FHF2): gene structure, expression and mapping to the Borjeson-Forssman-Lehmann syndrome region in Xq26 delineated by a duplication breakpoint in a BFLS-like patient. Hum Genet 104(1):56–63CrossRefGoogle Scholar
- Goetz R, Dover K, Laezza F, Shtraizent N, Huang X, Tchetchik D, Eliseenkova AV, Xu CF, Neubert TA, Ornitz DM, Goldfarb M, Mohammadi M (2009) Crystal structure of a fibroblast growth factor homologous factor (FHF) defines a conserved surface on FHFs for binding and modulation of voltage-gated sodium channels. J Biol Chem 284(26):17883–17896. https://doi.org/10.1074/jbc.M109.001842 CrossRefGoogle Scholar
- Goldfarb M, Schoorlemmer J, Williams A, Diwakar S, Wang Q, Huang X, Giza J, Tchetchik D, Kelley K, Vega A, Matthews G, Rossi P, Ornitz DM, D'Angelo E (2007) Fibroblast growth factor homologous factors control neuronal excitability through modulation of voltage-gated sodium channels. Neuron 55(3):449–463. https://doi.org/10.1016/j.neuron.2007.07.006 CrossRefGoogle Scholar
- Kettunen P, Furmanek T, Chaulagain R, Kvinnsland IH, Luukko K (2011) Developmentally regulated expression of intracellular Fgf11-13, hormone-like Fgf15 and canonical Fgf16, -17 and -20 mRNAs in the developing mouse molar tooth. Acta Odontol Scand 69(6):360–366. https://doi.org/10.3109/00016357.2011.568968 CrossRefGoogle Scholar
- Olsen SK, Garbi M, Zampieri N, Eliseenkova AV, Ornitz DM, Goldfarb M, Mohammadi M (2003) Fibroblast growth factor (FGF) homologous factors share structural but not functional homology with FGFs. J Biol Chem 278(36):34226–34236. https://doi.org/10.1074/jbc.M303183200
- Ornitz DM (2000) FGFs, heparan sulfate and FGFRs: complex interactions essential for development. Bioessays 22(2):108–112. https://doi.org/10.1002/(sici)1521-1878(200002)22:2<108::aid-bies2>3.0.co;2-m CrossRefGoogle Scholar
- Smallwood PM, Munoz-Sanjuan I, Tong P, Macke JP, Hendry SH, Gilbert DJ, Copeland NG, Jenkins NA, Nathans J (1996) Fibroblast growth factor (FGF) homologous factors: new members of the FGF family implicated in nervous system development. Proc Natl Acad Sci U S A 93(18):9850–9857CrossRefGoogle Scholar
- Wang C, Hennessey JA, Kirkton RD, Wang C, Graham V, Puranam RS, Rosenberg PB, Bursac N, Pitt GS (2011) Fibroblast growth factor homologous factor 13 regulates Na+ channels and conduction velocity in murine hearts. Circ Res 109(7):775–782. https://doi.org/10.1161/CIRCRESAHA.111.247957 CrossRefGoogle Scholar
- Zheng J, Wang S, Yang H, Chen Z, Huang S, Zhao T, Pan X, Fernig DG, Jiang C, Li X, Tian H (2017) Large-scale expression, purification of bioactive recombinant human FGF6 in E. coli and the mechanisms of its myocardial protection. Int J Pept Res Ther 24(1):105–115. https://doi.org/10.1007/s10989-017-9592-6 CrossRefGoogle Scholar