Expanded-bed adsorption immobilized-metal affinity chromatography |
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| Authors: | Tolner Berend Smith Lisa Begent Richard H J Chester Kerry A |
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| Institution: | 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 |
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| Abstract: | 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. |
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