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| Our research involves the use of chemical, biochemical and molecular biological approaches to study structure/function relationships of a family of enzymes known as glycosyltransferases. These enzymes catalyze the synthesis of biomolecules (i.e. glycoconjugates) that have several biological functions including acting as ligands for proteins that direct leukocyte trafficking. We have used site directed mutagenesis and other molecular biology approaches to identify the catalytic domain of these proteins and are in the process of identifying amino acid residues that form the substrate binding sites of the enzymes. Mass spectrometric analysis is being used, in conjunction with chemical modifications, to probe the post translational modifications that occur on the enzymes and to assist our molecular biology studies to identify the substrate binding sites. Junior and Senior level students in my research group are currently working on two types of projects and similar projects would be available for MARC students. The first project involves learning and applying molecular biology methods to modify the coding sequence of a glycosyltransferase by site directed mutagenesis. The modified sequence is prepared by PCR, cloned into an expression vector and sequenced to verify that the mutation has occurred, and that no other PCR-related modifications have been incorporated. The student learns molecular biology techniques, bacterial culture methods and general lab procedures from this experience. Subsequently, the students learn how to express the glycosyltransferase in a mammalian expression system. Thus, they learn cell culture, transfection, protein purification, SDS-PAGE and enzyme assay techniques. From the results obtained, the students learn about the importance of amino acid functional groups and how changing them can affect substrate binding and enzyme activity. The second project involves protein chemistry and mass spectrometry. For example, we recently determined the disulfide-bonding pattern in several glycosyltransferases. Some students were involved in the development and testing of the procedures required for this analysis and subsequently others students actually carried out the analysis on the glycosyltransferase. The students learn how to handle a small quantity of protein, how to denature, chemically modify and proteolytically digest the proteins. The also became familiar with reverse phase HPLC and electrospray mass spectrometry and how to use the mass spectrometer data system in conjunction with the protein data bases on the WWW to analyze and interpret their results.
Last modified July 10, 2002 |
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