TY - JOUR
T1 - Purification and characterization of Ras related protein, Rab5a from Tinospora cordifolia
AU - Amir, Mohd
AU - Wahiduzzaman,
AU - Dar, Mohammad Aasif
AU - Haque, Md Anzarul
AU - Islam, Asimul
AU - Ahmad, Faizan
AU - Hassan, Md Imtaiyaz
N1 - Funding Information:
We thank the Director of Herbarium garden Bhud kalan Haryana (Yamuna Nagar, Haryana). WZ, MAD are thankful to the ICMR and MAH is thankful to UGC for their fellowships. FA and MIH are thankful to the Indian Council of Medical Research and Department of Science and Technology for financial support.
Publisher Copyright:
© 2015 Elsevier B.V.
PY - 2016/1/1
Y1 - 2016/1/1
N2 - 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.
AB - 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.
KW - Chemical denaturation
KW - Protein purification
KW - Ras related protein
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U2 - 10.1016/j.ijbiomac.2015.10.077
DO - 10.1016/j.ijbiomac.2015.10.077
M3 - Article
C2 - 26517959
AN - SCOPUS:84951309919
SN - 0141-8130
VL - 82
SP - 471
EP - 479
JO - International Journal of Biological Macromolecules
JF - International Journal of Biological Macromolecules
ER -