Research Resource for Integrated Glycotechnology:

Kelley Moremen

Expression of Glycosylation Enzymes for Structural and Functional Studies

We are developing methods for the generation of sufficient quantities, and suitable forms of model protein targets for structural studies of protein-carbohydrate interactions by NMR, mass spectrometry, and X-ray diffraction. Methods that are directed toward the physical characterization of substrate/ligand binding sites require the isolation of large quantities (~100 mg) of unlabeled enzymes/lectins or smaller quantities (~10 mg) of isotope-enriched forms (¹⁵N-labeled) or selenomethionine-substituted forms of these proteins. We are investigating the methylotrophic yeast Pichia pastoris as a heterologous expression host for generating unlabeled and isotope-enriched forms of these proteins. This expression system has the advantage of generating recombinant glycoproteins that possess those post-translational modifications characteristic of eukaryotic organisms that are often required for biological activity and solubility of the expressed protein. We expect this system to also provide the framework for the expression, characterization, and analysis of other carbohydrate-binding proteins including other lectins, enzymes, and antibodies.

Development of host systems for recombinant protein expression.

One of the challenges in proteomics is the development of appropriate technologies for high throughput and high efficiency expression of proteins that require eukaryotic post-translational modifications for functional activity. Many proteins are not amenable to expression in bacterial systems, where isotope-enrichment or selenomethionine incorporation methods have been well established. Equivalent expression strategies in eukaryotic host systems have not been proven economical and, in many cases, high-level protein expression cannot be readily attained. Thus we would like to define the parameters for protein expression in eukaryotic cells using defined culture conditions that could provide high expression levels and isotope incorporation methods. We have chosen two commonly utilized expression hosts cell systems with the objective of defining parameters for high and versatile protein expression. The most studied of our expression host systems is Pichia pastoris, while an alternate expression strategy in human HEK293 cells is also being pursued.