Overexpression and Purification of Mitogenic and Metabolic Fibroblast Growth Factors.

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. © 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Citation

Phuc Phan, Shivakumar Sonnaila, Gaetane Ternier, Oshadi Edirisinghe, Patience Salvalina Okoto, Thallapuranam Krishnaswamy Suresh Kumar. Overexpression and Purification of Mitogenic and Metabolic Fibroblast Growth Factors. Methods in molecular biology (Clifton, N.J.). 2024;2762:151-181

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PMID: 38315365

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