Abstract
Fibroblast growth factors (FGFs) are proteins with a vast array of biological activity, such as cell development and repair, glucose and bile acid metabolisms, and wound healing. Due to their critical and diverse physiological functions, FGFs are believed to possess potential as therapeutic agents for many diseases and conditions that warrant further investigations. Thus, a simple, cost-efficient method to purify these biologically active signaling proteins is desirable. Herein, we introduce such techniques to purify FGFs that possess either high heparin-binding affinity or low to no heparin-binding affinity. This method takes advantage of the high affinity toward heparin sulfate from paracrine FGF1 to isolate the targeted protein. It also accounts for FGF members that have low heparin affinity, such as the metabolic FGFs, by introducing poly-histidine tags in the recombinant protein in combination with the immobilized metal affinity chromatography. Subsequently, the purified FGF products are separated from the other small protein by high-speed centrifugation. Products are then subjected to other biophysical experiments like SDS-PAGE, mass spectrometry, circular dichroism, intrinsic fluorescence, isothermal titration calorimetry, differential scanning calorimetry, and biological cell activity assay to confirm that the target proteins are purified with intact native conformation and no significant change in the intrinsic characteristics and biological activities.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Agrawal S (2020) Design and characterization of FGF-1 mutant (s) with increased stability and enhanced bioactivity. University of Arkansas
Agrawal S, Govind Kumar V, Gundampati RK, Moradi M, Kumar TKS (2021) Characterization of the structural forces governing the reversibility of the thermal unfolding of the human acidic fibroblast growth factor. Sci Rep 11:15579. https://doi.org/10.1038/s41598-021-95050-2
Benet-Pagès A, Orlik P, Strom TM, Lorenz-Depiereux B (2004) An FGF23 missense mutation causes familial tumoral calcinosis with hyperphosphatemia. Hum Mol Genet 14:385–390. https://doi.org/10.1093/hmg/ddi034
Bornhorst JA, Falke JJ (2000) Purification of proteins using polyhistidine affinity tags. Methods Enzymol 326:245–254. https://doi.org/10.1016/s0076-6879(00)26058-8
Carpenter TO et al (2018) Burosumab therapy in children with X-linked hypophosphatemia. New Engl J Med 378:1987–1998. https://doi.org/10.1056/nejmoa1714641
Crowe J, Döbeli H, Gentz R, Hochuli E, Stüber D, Henco K (1994) 6xHis-Ni-NTA chromatography as a superior technique in recombinant protein expression/purification. Methods Mol Biol 31:371–387. https://doi.org/10.1385/0-89603-258-2:371
Dolegowska K, Marchelek-Mysliwiec M, Nowosiad-Magda M, Slawinski M, Dolegowska B (2019) FGF19 subfamily members: FGF19 and FGF21. J Physiol Biochem 75:229–240. https://doi.org/10.1007/s13105-019-00675-7
Dunshee DR et al (2016) Fibroblast activation protein cleaves and inactivates fibroblast growth factor 21. J Biol Chem 291:5986–5996. https://doi.org/10.1074/jbc.M115.710582
Gaich G et al (2013) The effects of LY2405319, an FGF21 analog, in obese human subjects with type 2 diabetes. Cell Metab 18:333–340. https://doi.org/10.1016/j.cmet.2013.08.005
Gillum MP, Potthoff MJ (2016) FAP finds FGF21 easy to digest. Biochem J 473:1125. https://doi.org/10.1042/BCJ20160004
Ho BB, Bergwitz C (2021) FGF23 signalling and physiology. J Mol Endocrinol 66:R23–R32. https://doi.org/10.1530/JME-20-0178
Kerr R et al (2019) Design of a thrombin resistant human acidic fibroblast growth factor (hFGF1) variant that exhibits enhanced cell proliferation activity. Biochem Biophys Res Commun 518:191–196. https://doi.org/10.1016/j.bbrc.2019.08.029
Kharitonenkov A et al (2013) Rational design of a fibroblast growth factor 21-based clinical candidate, LY2405319. PLoS One 8:e58575. https://doi.org/10.1371/journal.pone.0058575
Lee S et al (2018) Structures of β-klotho reveal a ‘zip code’-like mechanism for endocrine FGF signalling. Nature 553:501–505. https://doi.org/10.1038/nature25010
Liu SH, Xiao Z, Mishra SK, Mitchell JC, Smith JC, Quarles LD, Petridis L (2022) Identification of small-molecule inhibitors of fibroblast growth factor 23 signaling via in silico hot spot prediction and molecular docking to α-Klotho. J Chem Inf Model 62:3627–3637. https://doi.org/10.1021/acs.jcim.2c00633
Lu Y, Feng JQ (2011) FGF23 in skeletal modeling and remodeling. Curr Osteoporos Rep 9:103–108. https://doi.org/10.1007/s11914-011-0053-4
Ornitz DM, Itoh N (2015) The fibroblast growth factor signaling pathway. Wiley Interdiscip Rev Dev Biol 4:215. https://doi.org/10.1002/wdev.176
Phan P, Saikia BB, Sonnaila S, Agrawal S, Alraawi Z, Kumar TKS, Iyer S (2021) The saga of endocrine FGFs. Cells 10:2418. https://doi.org/10.3390/cells10092418
Porath J (1992) Immobilized metal ion affinity chromatography. Protein Expr Purif 3:263–281. https://doi.org/10.1016/1046-5928(92)90001-d
Shimada T et al (2001) Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia. Proc Natl Acad Sci U S A 98:6500–6505. https://doi.org/10.1073/pnas.101545198
Shimada T et al (2002) Mutant FGF-23 responsible for autosomal dominant hypophosphatemic rickets is resistant to proteolytic cleavage and causes hypophosphatemia in vivo. Endocrinology 143:3179–3182. https://doi.org/10.1210/endo.143.8.8795
Somm E, Jornayvaz FR (2018) Fibroblast growth factor 15/19: From basic functions to therapeutic perspectives. Endocr Rev 39:960–989. https://doi.org/10.1210/er.2018-00134
Suzuki Y et al (2020) FGF23 contains two distinct high-affinity binding sites enabling bivalent interactions with α-Klotho. Proc Natl Acad Sci U S A 117:31800–31807. https://doi.org/10.1073/pnas.2018554117
Zhen EY, Jin Z, Ackermann BL, Thomas MK, Gutierrez JA (2016) Circulating FGF21 proteolytic processing mediated by fibroblast activation protein. Biochem J 473:605–614. https://doi.org/10.1042/BJ20151085
Zhu L et al (2021) Dynamic folding modulation generates FGF21 variant against diabetes. EMBO Rep 22:e51352. https://doi.org/10.15252/embr.202051352
Acknowledgments
We would like to thank the AIMRC NIH COBRE (P20GM139768), Department of Energy (DE-FG02-01ER15161), University of Arkansas Honors College for financial support. T.K.S.K. is the Mildred – Cooper Chair of Bioinformatics and would like to gratefully acknowledge this endowment grant for funding this research.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Phan, P., Sonnaila, S., Ternier, G., Edirisinghe, O., Okoto, P.S., Kumar, T.K.S. (2024). Overexpression and Purification of Mitogenic and Metabolic Fibroblast Growth Factors. In: Bradfute, S.B. (eds) Recombinant Glycoproteins. Methods in Molecular Biology, vol 2762. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3666-4_10
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3666-4_10
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3665-7
Online ISBN: 978-1-0716-3666-4
eBook Packages: Springer Protocols