title: Host selection as a downstream strategy: polyelectrolyte precipitation of beta-glucuronidase from plant extracts.
abstract: Host selection can be a strategy to simplify downstream processing for protein recovery. Advancing capabilities for using plants as hosts offers new host opportunities that have received only limited attention from a downstream processing perspective. Here, we investigated the potential of using a polycationic precipitating agent (polyethylenimine; PEI) to precipitate an acidic model protein (beta-glucuronidase; GUS) from aqueous plant extracts. To assess the potential of host selection to enhance the ease of recovery, the same procedure was applied to oilseed extracts of canola, corn (germ), and soy. For comparison, PEI precipitation of GUS was also evaluated from a crude bacterial fermentation broth. Two versions of the target protein were investigated--the wild-type enzyme (WTGUS) and a genetically engineered version containing 10 additional aspartates on each of the enzyme's four homologous subunits (GUSD10). It was found that canola was the most compatible expression host for use with this purification technique. GUS was completely precipitated from canola with the lowest dosage of PEI (30 mg PEI/g total protein), and over 80% of the initial WTGUS activity was recovered with 18-fold purification. Precipitation from soy gave yields over 90% for WTGUS but only 1.3-fold enrichment. Corn, although requiring the most PEI relative to total protein to precipitate (210 mg PEI/g total protein for 100% precipitation), gave intermediate results, with 81% recovery of WTGUS activity and a purification factor of 2.6. The addition of aspartate residues to the target protein did not enhance the selectivity of PEI precipitation in any of the systems tested. In fact, the additional charge reduced the ability to recover GUSD10 from the precipitate, resulting in lower yields and enrichment ratios compared to WTGUS. Compared to the bacterial host, plant systems provided lower polymer dosage requirements, higher yields of recoverable activity and greater purification factors.
authors.affiliation: Department of Chemical Engineering, 2114 Sweeney Hall, Iowa State University, Ames, IA 5001-2230, USA.
authors.forename: Todd J
authors.initials: TJ
authors.lastname: Menkhaus
authors.forename: Sara U
authors.initials: SU
authors.lastname: Eriksson
authors.forename: Paul B
authors.initials: PB
authors.lastname: Whitson
authors.forename: Charles E
authors.initials: CE
authors.lastname: Glatz
mesh_terms: D011232:Chemical Precipitation; D002853:Chromatography, Liquid / Q000379:methods; D004573:Electrolytes / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D005966:Glucuronidase / Q000235:genetics / Q000302:isolation & purification*; D010936:Plant Extracts / Q000737:chemistry*; D030821:Plants, Genetically Modified / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
chemical_list: D004573:Electrolytes; D010936:Plant Extracts; D011994:Recombinant Proteins; D005966:Glucuronidase