(taken from the applicants' Specific Aims) The goal of this project is to develop technology for generating sufficient quantities and suitable forms of model enzyme targets for kinetic, biophysical and structural studies on enzyme-substrate or enzyme-inhibitor interactions. The recombinant proteins generated in the proposed studies will be used to examine enzyme kinetic and thermodynamic parameters [enzyme kinetics, binding kinetics (Biacore), binding thermodynamics (ITC), substrate specificities and inhibitor profiles], biophysical characteristics and structural features among members of several glycosyltransferase families. The large quantities of recombinant enzymes generated will also provide sufficient starting materials for structural studies by NMR (Prestegard, Technology Project 4), mass (Orlando, Technology Project 3) and X-ray diffraction (Moreman and Wang, Technology Project 1). These studies will be focused on the interactions between enzymes and their carbohydrate substrates or inhibitors by taking advantage of substrates and substrate analogs synthesized in the Boons lab (Technology Project 2). ? ? Two independent strategies will be developed for recombinant protein expression that extend technologies developed in the previous funding period. The requirements for post-translational modifications and chaperonin systems in the production of these enzymes preclude the use of non-eukaryotic recombinant hosts for expression of many functional eukaryotic glycosylation enzymes. The applicants have therefore chosen two alternative eukaryotic expression strategies (Pichia pastoris and HEK293 cells) for recombinant enzyme production. Both strategies have previously proven effective for the production of recombinant mammalian glycosylhydrolases for structural and kinetic analyses in the Moreman lab.
The aims of the proposed studies extend on preliminary data for glycosidase and initial glycosyltransferase expression by (1) scale-up and isolation of >milligram quantities of model glycosyltransferases for kinetic, biophysical and structural studies; (2) development of strategies for 15N, 14C and SeMet labeling of recombinant proteins in Pichia and mammalian cells; (3) development of strategies for remodeling the glycosylation of recombinant proteins for analysis by biophysical and structural methods and (4) development of technologies for characterization of glycosylation enzymes by kinetic, biophysical and structural approaches. The applicants anticipate that the expression technologies developed in this Technology Project will also provide the framework for the characterization and analysis of other glycan-related proteins including other enzymes and lectins. ? ?
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