Purification and characterization of oligonucleotide binding (OB)-fold protein from medicinal plant Tinospora cordifolia

The oligonucleotide binding fold (OB-fold) is a small structural motif present in many proteins. It is originally named for its oligonucleotide or oligosaccharide binding properties. These proteins have been identified as essential for replication, recombination and repair of DNA. We have successfully purified a protein contains OB-fold from the stem of Tinospora cordifolia, a medicinal plants of north India. Stems were crushed and centrifuged, and fraction obtained at 60% ammonium sulphate was extensively dialyzed and applied to the weak anion exchange chromatography on Hi-Trap DEAE-FF in 50mM Tris-HCl buffer at pH 8.0. Eluted fractions were concentrated and applied to gel filtration column to get pure protein. We observed a single band of 20-kDa on SDS-PAGE. Finally, the protein was identified as OB-fold by MALDI-TOF. The purified OB-fold protein was characterized for its secondary structural elements using circular dichroism (CD) in the far-UV region. Generally the OB-fold has a characteristic feature as five-stranded beta-sheet coiled to form a closed beta- barrel. To estimate its chemical stability, guanidinium chloride-induced denaturation curve was followed by observing changes in the far-UV CD as a function of the denaturant concentration. Analysis of this denaturation curve gave values of 8.90±0.25kcalmol^-1 and 3.78±0.18M for δG_D° (Gibbs free energy change at 25°C) and C_m (midpoint of denaturation), respectively. To determine heat stability parameters of OB-fold protein, differential scanning calorimetry was performed. Calorimetric values of δG_D°, T_m (midpoint of denaturation), δH_m (enthalpy change at T_m), and δC_p (constant-pressure heat capacity change) are 9.05±0.27kcalmol^-1, 85.2±0,3°C, 105±4kcalmol^-1 and 1.6±0.08kcalmol^-1K^-1. This is the first report on the isolation, purification and characterization of OB-fold protein from a medicinal plant T. cordifolia.