Affinity Ligand for the Separation of Glycosylated Proteins from Non‐Glycosylated Proteins

Abstract

When studying glycosylated proteins, a method of separating glycosylated proteins from other proteins is crucial for purifying the sample. Affinity chromatography is currently performed most commonly for glycoprotein purification, through the use of boronic acid based, or lectin derived, ligands. However, more specific and economical ligands would be preferred. One such ligand described here is an affinity ligand that binds to glycosylated proteins and the monosaccharide α‐D‐mannopyranoside, synthesized through an Ugi‐multicomponent reaction. The ligand is composed of tryptamine, 1‐benzyl‐1H‐indole‐3‐carboxylic acid, isopropyl isocyanide, and an aldehyde‐functionalized Sepharose bead. The ligand's structure was selected via performance in a previously produced combinatorial library. For this work, after aldehyde functionalizing Sepharose beads and completing the Ugi reaction, the binding ability of the ligand is tested with gravity packed chromatography columns using glucose oxidase as the protein of interest. Then a Bradford assay is conducted in order to determine the amount of glycosylated protein (glucose oxidase) that bound to the affinity ligand during a positive and negative screening process. The ligand was initially synthesized and found to bind the glycoprotein glucose oxidase well in an inert buffer, with glycoprotein binding being reduced when a competitive ligand was added to the equilibrium buffer. Reproducibility of ligand synthesis and binding behavior is being assessed with multiple, separate ligand synthesis reactions and screens in order to determine optimal synthetic steps and assess the degree of sugar specificity in the ligands ability to bind the glycoprotein glucose oxidase.