Abstract:
Molecular sub-cloning and heterologous expression of the cDNA of reversibly glycosylated
protein from Cyamopsis tetragonoloba (CtRGP) was carried out in pET expression vector
(pET 29a (+) and the sub-cloned DNA was over-expressed in E. coli BL21 (DE3). The
recombinant protein purification was carried out by using immobilized metal ion
chromatography (IMAC). Molecular weight of purified protein was found to be
approximately 42 kDa. The purification was further confirmed by western blotting with anti-
RGP antibody. Biophysical characterization of CtRGP was done by analytical gel filtration,
MALDI-TOF/MS, peptide mass fingerprinting, circular dichroism (CD), fluorescence
spectroscopy (FS) and isothermal titration calorimetry (ITC). CtRGP protein was found to be
in tetrameric form. The molecular mass of CtRGP protein was found to be 42,123.21Da.
Peptide mass fingerprinting (PMF) analysis showed a total of 20 hits and most of these
proteins were the enzymes involved in mutase activity for plant cell wall synthesis. The CD
spectra of native CtRGP revealed the presence of 32.45% α-helix and 12.92% β-sheet.
Thermal and chemical denaturation of CtRGP protein showed high thermostability and
chemostability. The fluorescence study indicated that the tryptophan residue is located in the
hydrophobic core of the protein. The ITC studies with the substrate UDP-glucose and
inhibitors diisothiocyanostilbenedisulfonic acid (DIDS) and dithiobisnitrobenzoic acid
(DTNB) showed that purified CtRGP protein has a single binding site for its substrate and
inhibitors. The protein-ligands energetics revealed that the CtRGP protein has a strong,
moderate and weak binding affinities with inhibitor DIDS, substrate UDP-glucose and
inhibitor DTNB, respectively. In silico results showed that CtRGP is a stable protein and it
belongs to the RGP superfamily. The secondary structure prediction of the protein revealed
the presence of high helical content. Docking results showed that substrate UDP-glucose
interacts with the loop section of the CtRGP protein structure.
