title: Purification and characterization of Ras related protein, Rab5a from Tinospora cordifolia.
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.
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India.
authors.forename: Mohd
authors.initials: M
authors.lastname: Amir
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India.
authors.lastname: Wahiduzzaman
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India.
authors.forename: Mohammad Aasif
authors.initials: MA
authors.lastname: Dar
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India.
authors.forename: Md Anzarul
authors.initials: MA
authors.lastname: Haque
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India.
authors.forename: Asimul
authors.initials: A
authors.lastname: Islam
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India.
authors.forename: Faizan
authors.initials: F
authors.lastname: Ahmad
authors.affiliation: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India. Electronic address: mihassan@jmi.ac.in.
authors.forename: Md Imtaiyaz
authors.initials: MI
authors.lastname: Hassan
mesh_terms: D000595:Amino Acid Sequence; D002152:Calorimetry, Differential Scanning; D002850:Chromatography, Gel; D002942:Circular Dichroism; D008024:Ligands; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D011485:Protein Binding; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013816:Thermodynamics; D031610:Tinospora / Q000737:chemistry / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: D008024:Ligands; D010940:Plant Proteins; D020696:rab5 GTP-Binding Proteins
keywords: Chemical denaturation; Protein purification; Ras related protein