title: Mechanically stable porous cellulose media for affinity purification of family 9 cellulose-binding module-tagged fusion proteins.
abstract: A mechanically stable cellulose-based chromatography media was synthesized to permit inexpensive affinity purification of recombinant proteins containing the family 9 carbohydrate-binding module (CBM9) fused to either the N- or C-terminus of the target protein. A second-order response surface model was used to identify optimal concentrations of the primary reactants, epichlorohydrin and dimethyl sulfoxide (DMSO), required to cross-link the starting material, Perloza MT100, a compressible but inexpensive cellulose-based chromatography resin. This resulted in a mechanically stable cross-linked affinity chromatography media capable of operating at an order-of-magnitude higher linear velocity than permitted by unmodified MT100. Moments and Van Deemter analyses were used to show that rates of solute mass transfer within the column are largely unaffected by the cross-linking reaction, while the binding capacity decreased by 20% to 7.1 micromol of protein/g resin, a value superior to most commercial affinity chromatography media. In sharp contrast to MT100, the mechanical stability and purification performance of the cross-linked media are not diminished by scale-up or repeated column use.
authors.affiliation: The Michael Smith Laboratories, The University of British Columbia, Vancouver, BC V6T 1Z4, Canada.
authors.forename: Mojgan
authors.initials: M
authors.lastname: Kavoosi
authors.forename: Dexter
authors.initials: D
authors.lastname: Lam
authors.forename: Jenny
authors.initials: J
authors.lastname: Bryan
authors.forename: Douglas G
authors.initials: DG
authors.lastname: Kilburn
authors.forename: Charles A
authors.initials: CA
authors.lastname: Haynes
mesh_terms: D000327:Adsorption; D002482:Cellulose / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods*; D003432:Cross-Linking Reagents / Q000737:chemistry; D017743:Equipment Reuse; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*
chemical_list: D003432:Cross-Linking Reagents; D011993:Recombinant Fusion Proteins; D002482:Cellulose