title: The development, characterization, and demonstration of a novel strategy for purification of recombinant proteins expressed in plants.
abstract: Plants have attracted increasing attention as an expression platform for the production of pharmaceutical proteins due to its unlimited scalability and low cost potential. However, compared to other expression systems, plants accumulate relatively low levels of foreign proteins, thus necessitating the development of efficient systems for purification of foreign proteins from plant tissues. We have developed a novel strategy for purification of recombinant proteins expressed in plants, based on genetic fusion to soybean agglutinin (SBA), a homotetrameric lectin that binds to N-acetyl-D-galactosamine. Previously it was shown that high purity SBA could be recovered from soybean with an efficiency of greater than 90% following one-step purification using N-acetyl-D-galactosamine-agar columns. We constructed an SBA fusion protein containing the reporter green fluorescent protein (GFP) and transiently expressed it in N. benthamiana plants. We achieved over 2.5% of TSP accumulation in leaves of N. benthamiana. Confocal microscopic analysis demonstrated in vivo activity of the fused GFP partner. Importantly, high purity rSBA-GFP was recovered from crude leaf extract with ~90% yield via one-step purification on N-acetyl-D-galactosamine-agar columns, and the purified fusion protein was able to induce the agglutination of rabbit red blood cells. Combined with this, tetrameric assembly of the fusion protein was demonstrated via western blotting. In addition, rSBA-GFP retained its GFP signal on agglutinated red blood cells, demonstrating the feasibility of using rSBA-GFP for discrimination of cells that bear the ligand glycan on their surface. This work validates SBA as an effective affinity tag for simple and rapid purification of genetically fused proteins.
authors.affiliation: Department of Biology, University of Western Ontario, London, ON, Canada.
authors.forename: Reynald
authors.initials: R
authors.lastname: Tremblay
authors.forename: Hong
authors.initials: H
authors.lastname: Diao
authors.forename: Norm
authors.initials: N
authors.lastname: Huner
authors.forename: Anthony M
authors.initials: AM
authors.lastname: Jevnikar
authors.forename: Shengwu
authors.initials: S
authors.lastname: Ma
mesh_terms: D000116:Acetylgalactosamine / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism; D016960:Agrobacterium tumefaciens / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001709:Biotechnology / Q000379:methods*; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004903:Erythrocyte Aggregation; D018506:Gene Expression Regulation, Plant; D049452:Green Fluorescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D010936:Plant Extracts / Q000378:metabolism; D018515:Plant Leaves / Q000378:metabolism; D037121:Plant Lectins / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D030262:Soybean Proteins / Q000235:genetics / Q000378:metabolism*; D013025:Glycine max / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D010835:Phytohemagglutinins
chemical_list: D000116:Acetylgalactosamine; D000345:Affinity Labels; D049452:Green Fluorescent Proteins; D010936:Plant Extracts; D037121:Plant Lectins; D011993:Recombinant Fusion Proteins; D030262:Soybean Proteins; C028824:soybean lectin; D010835:Phytohemagglutinins