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Updated: Jul 31, 2026
Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow
Published on: January 8, 2014
Large-scale production, bacterial localization assessment and immobilized metal affinity chromatography purification
R Robert1, G Clofent-Sanchez, A Hocquellet
1CNRS UMR 5536, RMSB Résonance Magnétique des Systèmes Biologiques, Université Victor Ségalen, Bât. 4a, 146 rue Léo Saignat, 33076 Bordeaux, France. remi.robert@rmsb.u-bordeaux2.fr
Abstract:
Our objective was to investigate the Escherichia coli localization (such as supernatant, cytoplasm and inclusion bodies) of an anti-alphaIIb-beta3 (alphaIIbbeta3) scFv fragment referred to as scFv[EBB3] produced in batch fermentation. Immobilized metal affinity chromatography (IMAC) purification was performed on supernatant using expanded bed absorbed technology (EBA) and on sonicated cells in native conditions over an immobilized copper-ion affinity column. Inclusion bodies were solubilized before IMAC purification and the refolding procedure was performed on the column. The majority of scFv[EBB3] were present as inclusion bodies (55%), whereas 36% were found in the cytoplasm and only 9% secreted in the supernatant. The scFv activity was assessed by enzyme-linked immunosorbent assay (ELISA), flow cytometry and immunohistochemistry analyses performed on a thrombus induced in vivo on an atherosclerotic rabbit model.
