Expression and Purification of the Recombinant Kinase Domain of FGFR2b and Study of its Structural Changes Due to the Interaction with Gallic Acid

Document Type : Original Article (s)

Authors

1 Department of Biology, School of Science, Damghan Branch, Islamic Azad University, Damghan, Iran

2 Assistant Professor, Cellular and Molecular Research Center AND Department of Biochemistry and Genetics, School of Medicine, Qazvin University of Medical Sciences, Qazvin, Iran

3 Associate Professor, Department of Biophysics and Biotechnology, School of Paramedical Sciences AND Cellular and Molecular Research Centre, Qazvin University of Medical Sciences, Qazvin, Iran

4 Associate Professor, Cellular and Molecular Research Center AND Department of Biochemistry and Genetics, School of Medicine, Qazvin University of Medical Sciences, Qazvin, Iran

Abstract

Background: FGFR2b plays a significant role in cell signaling pathway, regulating several key biological processes including cellular differentiation and proliferation. Genetic alterations of the tyrosine kinase domain of FGFR2b, such as point mutations, occur in breast, ovarian and prostate cancer. This study aimed to express and zepurify the human FGFR2b kinase domain and to analyze its structural changes upon interaction with Gallic acid (GA).Methods: Expression of recombinant protein was induced with 1mM IPTG at 37 ºC and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein was purified via affinity chromatography and the protein sample was dialyzed and then used to be analyzed via SDS-PAGE. Chemical denaturation and intrinsic fluorescence spectra of the purified proteins were carried out via adding different concentrations of Gallic acid.Findings: Comparison between pre- and post-induction samples via SDS-PAGE analysis showed that the expressed protein was soluble at 20 ºC. Additionally, its purity was confirmed. The intrinsic fluorescence spectra of kinase domain in the presence of Gallic acid showed an increase in fluorescence intensity and maximum emission wavelength.Conclusion: Regarding to the results, the recombinant kinase domain of FGFR2b (38 kDa) was expressed, solubilized and purified. Changing in tertiary structural kinase domain reflects a conformational change within the protein that is important for the biological function of FGFR2b.

Keywords

References

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Journal of Isfahan Medical School
Volume 33, Issue 362 - Serial Number 362
November and December 2016
Pages 2143-2151

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History

  • Receive Date: 26 August 2015
  • Revise Date: 01 November 2024
  • Accept Date: 06 April 2022

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