title: Preparation of affinity membranes using thermally induced phase separation for one-step purification of recombinant proteins.
abstract: We synthesized several surfactant-like ligands and prepared affinity membranes by introducing them into porous polymeric membranes using the thermally induced phase separation method. The ligands (nitrilotriacetate, iminodiacetate, and glutathione) were successfully displayed on the surfaces of cellulose diacetate membranes. Membranes functionalized with nitrilotriacetate and glutathione captured and released hexahistidine-tagged enhanced green fluorescent protein (His-tag GFP) and glutathione S-transferase (GST) selectively under appropriate conditions. The affinity membranes also enabled highly selective purification of target proteins (GFP and GST) from cell lysates. The protein-binding capacity was 15 μg/cm(2) for His-tag GFP and 13 μg/cm(2) for GST. The application-specific membranes described in this work will aid high-throughput screening and high-throughput analysis of recombinant proteins.
authors.affiliation: Department of Chemical Science and Engineering, Kobe University, Kobe 657-8501, Japan.
authors.forename: Takafumi
authors.initials: T
authors.lastname: Honjo
authors.forename: Kazuki
authors.initials: K
authors.lastname: Hoe
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Tabayashi
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Tanaka
authors.forename: Josui
authors.initials: J
authors.lastname: Shimada
authors.forename: Masahiro
authors.initials: M
authors.lastname: Goto
authors.forename: Hideto
authors.initials: H
authors.lastname: Matsuyama
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Maruyama
mesh_terms: D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000737:chemistry; D008024:Ligands; D008567:Membranes, Artificial; D008855:Microscopy, Electron, Scanning; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013501:Surface-Active Agents / Q000737:chemistry; D013696:Temperature; D013997:Time Factors
chemical_list: D008024:Ligands; D008567:Membranes, Artificial; D011994:Recombinant Proteins; D013501:Surface-Active Agents; D005978:Glutathione