369831 Lectin-Mediated Affinity Precipitation Affords High-Yield Recovery of Fucosylated Proteins from Plant Crude Extract

Monday, November 17, 2014: 9:48 AM
205 (Hilton Atlanta)
Lindsay Arnold, Chemical and Biomolecular Engineering, Georgia Tech, ATLANTA, GA

Effective methods for isolation and purification of glycoproteins and other glycoconjugates are important to biopharmaceutical industry and diagnostic industry. They are also critical to an emerging  field of glycoproteomics aimed to understand structure and function relationships of glycans. Previously, we reported development of thermal-responsive affinity ligands by fusing elastin like polymer (ELP) to a bacterial lectin. In this work, we apply the affinity ligand in an affinity precipitation process to isolate fucosylated soybean peroxidase (SBP) from a dilute crude plant extract. Under optimal conditions, one step binding and precipitation resulted in >95% recovery yield directly from crude extract and a 22.7 fold purification. The SBP isolated using this affinity precipitation meets or exceeds the quality specifications of reagent grade products by Sigma. We showed that the recovery yield had a strong dependence on the molar ratio of ligand to target fucosylated protein, with a ratio of three being optimal, which could be predicted based on the total fucose content per protein molecule and the number of binding site per ligand molecule. The optimal condition for elution of target protein was determined and a relatively high concentration of cognate sugar for the lectin (1mM fucose) was needed for effective elution (≥80%). To regenerate the ligand, fucose bound at the bind sites was washed off with a pH buffer containing no fucose. This simple wash process allowed the ligand to regain much of the binding ability. However, a gradual decline of binding ability was observed after each reuse. In spite of the decline, we demonstrated that the ligand could be used for 10 times, giving an averaged 80% isolation yield based on initial input of soybean peroxidase. Taken together, an effective method of affinity precipitation was developed, which could be used to enrich a low abundant target glycoprotein from a complex mixture with a high recovery yield. Although the method is only shown here for a fucosylated protein, it is conceivable that this method of purification can be extended to other glycoproteins by developing thermal responsive ligands with lectins specific to sugars other than fucose. Preliminary results of multi-lectin affinity precipitation (M-LAP) show promise in demonstrating a platform for expansion of these lectin fusions to target various sugars.  This enables further fractionation of various glycoprofiles from a range of samples.

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