Glycosylation is an important modification in proteins in that it will signal for proper folding of proteins. It also has several important roles in the cell for example structural roles like aiding in stability of proteins; they act as recognition epitopes of the proteins that they are attached to. Oligosaccharides on protein on cell surface also act as receptors of viruses, bacteria and toxins thus acting as the entry point of harmful agents into the body. Therefore the study of the structure of these compounds is important in understanding their roles and functions. To mimic this type of modification PEGylation has been used in therapeutic proteins. PEGylation helps mainly in increasing the half-life of these compounds in the body and also increase the solubility and stability of protein drugs.
In this research analytical methods were used to determine the glycans structure on recombinant glycoprotein CD24. In chapter 2, the average structures of glycans on CD24 were determined by the use of MALDI-TOF-MS after the glycans were released with chemicals means and enzymatic means. The result showed the presence of both N- and O-glycans with the major compound being the O-glycans. The O-glycans that mostly expressed was found to be the sialyl T-antigen. To determine the glycosylation sites on the protein one N-glycosylation site on the fusion portion was determined on the amino peptide, EEQYNSTYR after trypsin digestion.
In chapter four, the glycosylation study of N-glycans from a recombinant protein TNFR-Fc expressed in the plant Camelina sativa was carried. The N-glycans were released with PNGase A and PNGase F. The results revealed the presence of plant-like glycans due to the presence of Xylose and α-1,3 Fucose and animal-like N-glycans.
In the last chapter a variant of paraoxonase 1 -a catalytic bioscavenger against organophosphate compounds was expressed in E. coli and PEGylated with a 28 kDa NHS activated mPEG. PEGylation is an important protein modification in improving its solubility and serum half-life. After the PEGylation, the PEGylated protein was purified by anion exchange chromatography and the kinetic studies for this enzyme against paraoxon carried out. The result revealed a reduction of the Kcat/Km values when compared to the unPEGylated enzyme.