Purification and characterization of Ras related protein, Rab5a from Tinospora cordifolia

Mohd Amir, Wahiduzzaman, Mohammad Aasif Dar, Md Anzarul Haque, Asimul Islam, Faizan Ahmad, Md Imtaiyaz Hassan

Research output: Contribution to journalArticlepeer-review

Abstract

Ras related protein (Rab5a) is one of the most important member of the Rab family which regulates the early endosome fusion in endocytosis, and it also helps in the regulation of the budding process. Here, for the first time we report a simple and reproducible method for the purification of the Rab5a from a medicinal plant Tinospora cordifolia. We have used weak cation-exchange (CM-Sepharose-FF) followed by gel-filtration chromatography. A purified protein of 22-kDa was observed on SDS-PAGE which was identified as Rab5a using MALDI-TOF/MS. Our purification procedure is fast and simple with high yield. The purified protein was characterized using circular dichroism for the measurement of secondary structure followed by GdmCl- and urea-induced denaturation to calculate the values of Gibbs free energy change (δGD), δGD°, midpoint of the denaturation Cm, i.e. molar GdmCl [GdmCl] and molar urea [Urea] concentration at which δGD=0; and m, the slope (=∂δGD/∂[d]) values. Furthermore, thermodynamic properties of Rab5a were also measured by differential scanning calorimeter. Here, using isothermal calorimeteric measurements we further showed that Rab5a binds with the GTP. This is a first report on the purification and biophysical characterization of Rab5a protein from T. cordifolia.

Original languageEnglish (US)
Pages (from-to)471-479
Number of pages9
JournalInternational Journal of Biological Macromolecules
Volume82
DOIs
StatePublished - Jan 1 2016
Externally publishedYes

Keywords

  • Chemical denaturation
  • Protein purification
  • Ras related protein

ASJC Scopus subject areas

  • Molecular Biology
  • Structural Biology
  • Biochemistry

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