Large-scale production, bacterial localization assessment and immobilized metal affinity chromatography purification of a human single-chain Fv antibody against alphaIIb-beta3 integrin.
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.
Robert
R
R
CNRS 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
Clofent-Sanchez
G
G
Hocquellet
A
A
Jacobin-Valat
M-J
MJ
Daret
D
D
Noubhani
A M
AM
Santarelli
X
X
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D050197:Atherosclerosis / Q000276:immunology; D002853:Chromatography, Liquid; D004195:Disease Models, Animal; D004926:Escherichia coli / Q000737:chemistry / Q000254:growth & development*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D002479:Inclusion Bodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000276:immunology; D017510:Protein Folding; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D013927:Thrombosis / Q000139:chemically induced / Q000276:immunology
D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011993:Recombinant Fusion Proteins