title: Quantitative evaluation of E1 endoglucanase recovery from tobacco leaves using the vacuum infiltration-centrifugation method.
abstract: As a production platform for recombinant proteins, plant leaf tissue has many advantages, but commercialization of this technology has been hindered by high recovery and purification costs. Vacuum infiltration-centrifugation (VI-C) is a technique to obtain extracellularly-targeted products from the apoplast wash fluid (AWF). Because of its selective recovery of secreted proteins without homogenizing the whole tissue, VI-C can potentially reduce downstream production costs. Lab scale experiments were conducted to quantitatively evaluate the VI-C method and compared to homogenization techniques in terms of product purity, concentration, and other desirable characteristics. From agroinfiltrated Nicotiana benthamiana leaves, up to 81% of a truncated version of E1 endoglucanase from Acidothermus cellulolyticus was recovered with VI-C versus homogenate extraction, and average purity and concentration increases of 4.2-fold and 3.1-fold, respectively, were observed. Formulas were developed to predict recovery yields of secreted protein obtained by performing multiple rounds of VI-C on the same leaf tissue. From this, it was determined that three rounds of VI-C recovered 97% of the total active recombinant protein accessible to the VI-C procedure. The results suggest that AWF recovery is an efficient process that could reduce downstream processing steps and costs for plant-made recombinant proteins.
authors: Department of Chemical Engineering and Materials Science, University of California at Davis, 1 Shields Avenue, Davis, CA 95616, USA.
authors: Nathaniel J
authors: NJ
authors: Kingsbury
authors: Department of Chemical Engineering and Materials Science, University of California at Davis, 1 Shields Avenue, Davis, CA 95616, USA.
authors: Karen A
authors: KA
authors: McDonald
mesh_terms: D000192:Actinomycetales / Q000201:enzymology*; D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry / Q000302:isolation & purification*; D002498:Centrifugation / Q000379:methods*; D007203:Indigo Carmine / Q000378:metabolism; D009195:Peroxidase / Q000302:isolation & purification; D018515:Plant Leaves / Q000378:metabolism*; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D014618:Vacuum
chemical_list: D011994:Recombinant Proteins; D007203:Indigo Carmine; D009195:Peroxidase; D002480:Cellulase