Production of Recombinant Glycoproteins in Mammalian Cells for Structural and Biochemical Studies

A platform for recombinant expression of mammalian glycoproteins has been developed in the Moremen lab for biochemical and structural studies on glycosylation enzymes and glycan binding proteins. Successful production of mammalian glycosylation enzymes in the Moremen lab led to the expanded development of a large-scale project for the expression of all mammalian glycosylation enzymes through the Repository of Glycan-related Expression Constructs as a supplement to the Resource for Integrated Glycotechnology in the last funding cycle. This project is a collaboration between the Moremen lab, and the laboratories of Joshua LaBaer (Arizona State University) and Don Jarvis (University of Wyoming). Constructs encoding the soluble catalytic domains of most human glycosyltransferases, glycoside hydrolases, sulfotransferases, and other glycan modifying enzymes have been generated for expression in both insect cells and mammalian cells. Representative enzymes are being generated in multi-milligram quantities for enzymatic characterization, structural studies, chemo-enzymatic glycan synthesis using protein tagging strategies that facilitate stable expression, purification, quantitation, and subsequent tag removal for structural studies. The technology developed for mammalian expression has also been adapted for metabolic labeling of glycoproteins with NMR-active isotopes and insertion of paramagnetic tags to facilitate NMR structural studies. This platform is also being applied for the recombinant expression of numerous glycan binding studies in the Resource Driving Biomedical Projects and Collaborations.