Type of Document	Dissertation
Author	Walton, Wendy Jean
URN	etd-01062006-165049
Title	Isotopic Enrichment and Receptor-Binding Analysis of Insect and Lec1 Cell Expressed Avian Thy-1
Degree	Doctor of Philosophy
Department	Molecular Biophysics, Institute of
Advisory Committee	Advisor Name Title Thomas Keller III Committee Chair Alan Marshall Committee Member Hong Li Committee Member Richard Hyson Committee Member Tim Logan Committee Member
Keywords	Isotopic Enrichment NMR Receptor-Binding Analysis Thy-1 Glycoprotein Structure
Date of Defense	2005-12-09
Availability	unrestricted
Abstract Over half of the proteins found in nature are anticipated to be glycosylated, yet less than 2% of the protein structures in the protein data bank are glycoproteins. Clearly, we are behind in our structural understanding of glycoproteins. One reason for the lack of structural information for glycoproteins is due to their oligosaccharide heterogeneity. The two main methods for determining detailed three-dimensional protein structures are X-ray crystallography and nuclear magnetic resonance. Oligosaccharide heterogeneity hinders crystallization for X-ray crystallography analysis and complicates resonance assignment of nuclear magnetic resonance data. Another challenge is expressing high enough yields from eukaryotic cells for X-ray crystallography and nuclear magnetic resonance analysis. Additionally, unlike bacteria, eukaryotic cell lines typically cannot synthesize amino acids from glucose and ammonium chloride. Thus, eukaryotic media must contain amino acids for cell growth and protein production. Structural analysis of glycoproteins by nuclear magnetic resonance requires that the amino acids are labeled with 13C- and 15N and that the oligosaccharides are labeled with 13C. Media containing 13C- and 15N-labeled amino acids is extremely expensive. This dissertation explains methods that address each of these challenges utilizing the glycoprotein Thy-1. An economic approach for labeling recombinant protein expressed from insect cells for NMR is explained. A method for labeling oligosaccharides on glycoproteins expressed from mammalian cells is also explained. Changes in previously established expression and purification protocols for Thy-1 derived from Lec1 cells, which produce relatively homogeneous glycoforms, have increased yields to levels suitable for nuclear magnetic resonance studies. In addition, the biological relevancy of recombinant Thy-1, which is expressed with truncated oligosaccharides and without it’s glycosylphosphatidylinositol anchor, was confirmed by receptor-binding analysis.
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