title: Synthesis and performance of megaporous immobilized metal-ion affinity cryogels for recombinant protein capture and purification.
abstract: Megaporous cryogels with metal-ion affinity functionality, which possess enhanced protein-binding ability, were synthesized and their properties were investigated. These highly porous materials (pore sizes up to 100 μm) allowed the direct capture of a recombinant His(6)-tagged protein from a partially clarified extract. The total ligand density of the material was found to be 770 μmol/g. Application of a partially clarified cell extract in order to recover a His(6)-tagged protein (NAD(P)H-dependent 2-cyclohexen-1-one-reductase) yielded 12 mg of highly purified recombinant product per gram of adsorbent. Increased dynamic binding capacities were observed upon larger degrees of grafting, although some reduction in the quality of the system hydrodynamics was also observed. Nevertheless, these immobilized metal-ion affinity cryogels show potential for a convenient single-step purification of recombinant proteins from raw cell extracts without the need for laborious pre-chromatographic sample clean-up procedures.
authors.affiliation: Downstream Bioprocessing Laboratory, School of Engineering and Science, Jacobs University, Campus Ring 1, D-28759 Bremen, Germany.
authors.forename: Noor Shad
authors.initials: NS
authors.lastname: Bibi
authors.forename: Naveen Kumar
authors.initials: NK
authors.lastname: Singh
authors.forename: Roy N
authors.initials: RN
authors.lastname: Dsouza
authors.forename: Muhammad
authors.initials: M
authors.lastname: Aasim
authors.forename: Marcelo
authors.initials: M
authors.lastname: Fernández-Lahore
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D059985:Cryogels / Q000138:chemical synthesis* / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004852:Epoxy Compounds / Q000737:chemistry; D057446:Hydrodynamics; D007098:Imino Acids / Q000737:chemistry; D008670:Metals / Q000737:chemistry*; D008855:Microscopy, Electron, Scanning; D016062:Porosity; D011312:Pressure; D011994:Recombinant Proteins / Q000302:isolation & purification*; D012212:Rheology
chemical_list: D059985:Cryogels; D004852:Epoxy Compounds; D007098:Imino Acids; D008670:Metals; D011994:Recombinant Proteins; C008109:iminodiacetic acid