title: Capture of a recombinant protein from unclarified canola extract using streamline expanded bed anion exchange.
abstract: The feasibility of applying expanded bed adsorption technology to recombinant protein recovery from extracts of transgenic canola (rapeseed) was assessed. The extraction step results in a suspension of high solids content that is difficult to clarify. The coarse portion of the solids can be removed easily, and our aim was to operate the expanded bed in the presence of the recalcitrant particulates. Recombinant beta-glucuronidase (rGUS) produced in transgenic canola seed was the model system. Diethylaminoethyl (DEAE) and Streamline DEAE resin exhibited similar binding and elution properties for both rGUS and native canola proteins. More than 95% of native canola proteins did not bind to DEAE resins at pH 7.5, whereas the bound proteins were fractionated by two-step salt elution into two groups with the first peak, containing 70% of total bound proteins, at 20 mS/cm, followed by elution of rGUS at 50 mS/cm. The adsorption isotherm was only slightly influenced by the presence of up to 14 mg solids/mL extract; C(m) and K(d) changed by -1% and +39%, respectively. Bed expansion was semiquantitatively predictable from physical properties of the fluid together with Stokes's law and the Richardson-Zaki correlation for both clarified and partially clarified extracts. The presence of 1.4% solids did not change rGUS breakthrough behavior of the expanded bed; however, a small difference between expanded bed and packed bed was observed early in the sample loading stage, during which bed expansion adjusts. Canola solids moved through the column in approximately plug flow with no detriment to bed stability. Seventy-two percent recovery of 34-fold purified rGUS was obtained after initial loading of 1.4% (w/w) solids extract to 25% breakthrough.
authors.affiliation: Chemical Engineering Department, 2114 Sweeney Hall, Iowa State University, Ames, Iowa 50011-2230, USA.
authors.forename: Yun
authors.initials: Y
authors.lastname: Bai
authors.forename: Charles E
authors.initials: CE
authors.lastname: Glatz
mesh_terms: D000327:Adsorption; D029741:Brassica rapa / Q000235:genetics / Q000378:metabolism*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004983:Ethanolamines / Q000737:chemistry; D005240:Feasibility Studies; D005966:Glucuronidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D007475:Ion Exchange Resins / Q000737:chemistry*; D010936:Plant Extracts / Q000737:chemistry / Q000302:isolation & purification*; D030821:Plants, Genetically Modified / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012639:Seeds / Q000235:genetics / Q000378:metabolism; D012685:Sepharose / Q000737:chemistry
chemical_list: D004983:Ethanolamines; D007475:Ion Exchange Resins; D010936:Plant Extracts; D011994:Recombinant Proteins; D012685:Sepharose; D005966:Glucuronidase; C007369:2-diethylaminoethanol