title: Comparison of histidine-tag capture chemistries for purification following chemical extraction.
abstract: The purification of a 6x-histidine tagged viral coat protein (L1) in expanded mode directly following chemical extraction from the cytoplasm of Escherichia coli HMS174(DE3) is investigated. Chelating adsorbents based on the ligands iminodiacetic acid (IDA) and nitrilotriacetic acid, using chelated metal ions Ni2+ and Cu2+, were compared. The use of Ni2+-IDA resulted in a high purification factor (9.7) and moderate recovery yield (58%). However, the eluted fractions had an overall L1 purity less than 50% and were therefore significantly contaminated with other host proteins. In batch tests, Cu2+-IDA was found to be superior to all other combinations as it was characterised by higher binding capacities and faster adsorption kinetics. A subsequent immobilised metal affinity chromatography-expanded bed adsorption experiment using Cu2+-IDA resulted in a higher L1 purification factor (20), recovery yield (71%) and purity (89%). The process presented here combines direct chemical extraction with expanded bed recovery. It is simpler than traditional methods, and should find more widespread application in the recovery of inclusion body proteins. Robust pseudo-affinity ligands such as metal chelates show potential for selective primary recovery of unfolded proteins, and could be used for further processing such as on-column refolding.
authors.affiliation: Department of Chemical Engineering, University of Cambridge, UK.
authors.forename: Woo-Seok
authors.initials: WS
authors.lastname: Choe
authors.forename: Robert H
authors.initials: RH
authors.lastname: Clemmitt
authors.forename: Howard A
authors.initials: HA
authors.lastname: Chase
authors.forename: Anton P J
authors.initials: AP
authors.lastname: Middelberg
mesh_terms: D000327:Adsorption; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D006639:Histidine / Q000737:chemistry*; D007700:Kinetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D006639:Histidine