Large-scale production, bacterial localization assessment and immobilized metal affinity chromatography purification of a human single-chain Fv antibody against alphaIIb-beta3 integrin

Remy Robert, G Clofent-Sanchez, A Hocquellet, Marie-Josee Jacobin-Valat, D Daret, A M Noubhani, X Santarelli

Research output: Contribution to journalArticleResearchpeer-review

13 Citations (Scopus)

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.
Original languageEnglish
Pages (from-to)51 - 59
Number of pages9
JournalInternational Journal of Biological Macromolecules
Volume39
Issue number1-3
DOIs
Publication statusPublished - 2006
Externally publishedYes

Cite this