One-step non-chromatography purification of a low abundant fucosylated protein from complex plant crude extract

Abstract

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. In this work, we applied the newly-developed affinity ligand, a fusion protein of elastic like polymer (ELP) and a bacterial lectin, in an affinity precipitation process to purify soybean peroxidase (SBP) based on the presence of fucoseon the protein surface. We addressed, in particular, the challenge of purifying a low abundant protein from a complex dilute crude plant extract. The novel affinity precipitation developed in this work was very promising. One step binding and precipitation resulted in >95% recovery yield directly from crude extract and a 22.7 fold purification, giving a specific activity of 420 U/mg. 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 giving nearly full recovery, which could be predicted based on the total fucose content per protein molecule and the number of binding site per ligand molecule. We additionally developed a method of ligand regeneration and investigated its reuse. A simple wash with pH buffer was shown to be effective to regenerate the binding capacity for the ligand, and 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. The high selectivity for fucosylated protein and its ease of operation make this method particularly useful for purification of low abundant glycoprotein from natural sources. This work establishes a non-chromatography glycoform-specific purification method and extends the useful ELP-based affinity precipitation to glycoproteins.