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Publication

Expression and characterization of the membrane proteins tcr alpha WT and gamma-sarcolgycan with the quantification of in vitro glycosylated gamma-sarcoglycan

Harris, Michael Shaun
Abstract
Membrane proteins are a continually growing area of research in science as they are prevalent in a multitude of different disease states in humans. Membrane proteins are responsible for different biological processes in the cell such as cell-cell interactions, signal transduction, and transportation. However, very little is known about their structure and function because of their difficulty to work with them. Not only are membrane proteins difficult to express in high quantities, but they also contain highly hydrophobic portions which require detergents for solubilization to study these proteins. Expressing and purifying these proteins are only the first part in studying them, as most of them have post-translational modifications that can lead to structural and functional changes. N-linked glycosylation is a post-translational modification that can play a major role in the structural and functional changes of these membrane proteins by the attachment of a sugar and forming a glycoprotein. Therefore, using a novel in vitro technique, a chemoenzymatic reaction using an N-glycosyltransferase to attach a sugar moiety at a consensus sequence of Asn-X-Ser/Thr was performed. The attachment of the sugar moiety can influence physiological and biological properties of the protein by affecting their folding, modulating interactions with other biomolecules, and modifying their functions at the cellular level. We are specifically interested in the properties of membrane glycoproteins, which are key components in several different disease states. Therefore, gamma-Sarcoglycan and TCR alpha are the proteins being studied because they are linked to different disease states in humans and contain the consensus sequence for N-linked glycosylation. The ability to express and purify these proteins will be used to elucidate their structures through NMR spectroscopy and eventual determine changes in the proteins caused by glycosylation.
Date
2022-05