title: Purification of plant-derived antibodies through direct immobilization of affinity ligands on cellulose.
abstract: Plants possess enormous potential as factories for the large scale production of therapeutic reagents such as recombinant proteins and antibodies. A major factor limiting commercial advances of plant-derived pharmaceuticals is the cost and inefficiency of purification. As a model system, we have developed a simple yet robust method for immobilizing affinity capture ligands onto solid supports by interfacing the secreted expression and coupling of a chimeric fusion protein in Pichia pastoris to microcrystalline cellulose in a single step. The fusion protein, which consisted of antibody-binding proteins L and G fused to a cellulose-binding domain (LG-CBD), was tethered directly onto cellulose resins added to P. pastoris cultures and subsequently used for antibody purification. Both the antibody-binding protein L and protein G domains were functional, as demonstrated by the ability of cellulose-immobilized LG-CBD to purify both a scFv antibody fragment from yeast and a human IgG1 monoclonal antibody from transgenic tobacco. Furthermore, combining two P. pastoris strains expressing LG-CBD and scFv with CP-102 cellulose in a single culture allowed for easy recovery of biologically active scFv. Direct immobilization of affinity purification ligands, such as LG-CBD, onto inexpensive support matrices such as cellulose is an effective method for the generation of functional, single-use antibody purification reagents. Straightforward preparation of purification reagents will help make antibody purification from genetically modified crop plants feasible and address one of the major bottlenecks facing commercialization of plant-derived pharmaceuticals.
authors.affiliation: School of Environmental Sciences, University of Guelph, Ontario, Canada.
authors.forename: Greg
authors.initials: G
authors.lastname: Hussack
authors.forename: Brittany M
authors.initials: BM
authors.lastname: Grohs
authors.forename: Kurt C
authors.initials: KC
authors.lastname: Almquist
authors.forename: Michael D
authors.initials: MD
authors.lastname: McLean
authors.forename: Raja
authors.initials: R
authors.lastname: Ghosh
authors.forename: J Christopher
authors.initials: JC
authors.lastname: Hall
mesh_terms: D000906:Antibodies / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002352:Carrier Proteins; D002482:Cellulose / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism
chemical_list: D000906:Antibodies; D002352:Carrier Proteins; D011993:Recombinant Fusion Proteins; D002482:Cellulose; C109691:microcrystalline cellulose