Please use this identifier to cite or link to this item: http://localhost:8081/xmlui/handle/123456789/14520
Authors: Kumari, Shilpi
Keywords: Molecular Sub-Cloning;Heterologous Epression;Peptide Mass Fingerprinting;cCrcular Dichroism
Issue Date: Dec-2014
Publisher: Dept. of Biotechnology iit Roorkee
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.
metadata.dc.type: Thesis
Appears in Collections:DOCTORAL THESES (Bio.)

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