title: Novel thermo-responsive fucose binding ligands for glycoprotein purification by affinity precipitation.
abstract: Novel thermo-responsive affinity sugar binders were developed by fusing a bacterial fucose lectin with a thermo-responsive polypeptide. These designer affinity ligand fusions were produced using an Escherichia coli system capable of extracellular secretion of recombinant proteins and were isolated with a high recovery yield (95%) directly from growth medium by Inverse Temperature Cycling (ITC). With horse radish peroxidase (HRP) as a model protein, we demonstrate here that the designer thermo-responsive ligands are capable of interacting with glycans on a glycoprotein, a property that was used to develop a novel affinity precipitation method for glycoprotein purification. The method, requiring only simple process steps, affords full recovery of a target glycoprotein, and is effective at a target glycoprotein concentration as low as 1.4 pM in the presence of large amounts of contaminants. By developing other sugar binders in the similar fashion, the method should be highly useful for glycoprotein purification and detection.
authors.forename: Lindsay
authors.initials: L
authors.lastname: Arnold
authors.forename: Rachel
authors.initials: R
authors.lastname: Chen
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli; D005590:Fractional Precipitation / Q000379:methods*; D005643:Fucose / Q000378:metabolism*; D015870:Gene Expression; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D037102:Lectins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D006023:Glycoproteins; D037102:Lectins; D011994:Recombinant Proteins; D005643:Fucose