Expanded-bed adsorption immobilized-metal affinity chromatography.
The protocol describes a method for capture of secreted hexahistidine-tagged proteins using expanded-bed adsorption immobilized-metal affinity chromatography. The starting material for the procedure is any crude feedstock that contains a histidine (His)-tagged target protein. The protocol is exemplified using unclarified broth from Pichia pastoris fermentation as feedstock. The protocol can be used for laboratory studies or as part of a process for production of recombinant biotherapeutics to standards of good manufacturing practice. It takes approximately 5 h to purify proteins from 10 liters of feedstock and a further 5-6 h to sterilize and regenerate the column.
Tolner
Berend
B
Department of Oncology, Royal Free and University College Medical School, University College London, Rowland Hill Street, London NW3 2PF, UK. b.tolner@ucl.ac.uk
Smith
Lisa
L
Begent
Richard H J
RH
Chester
Kerry A
KA
D000327:Adsorption; D002846:Chromatography, Affinity / Q000295:instrumentation* / Q000379:methods*; D006639:Histidine; D008670:Metals; D009842:Oligopeptides; D011506:Proteins / Q000302:isolation & purification*
C471213:His-His-His-His-His-His; D008670:Metals; D009842:Oligopeptides; D011506:Proteins; D006639:Histidine