Improvement in production and purification bioprocesses of bacterially expressed anti-alphaIIbbeta3 human single-chain FV antibodies.
Production of anti-alphaIIbbeta3 (anti-alphaIIbbeta3)-binding single-chain FV (scFv) fragments obtained from combinatorial libraries of IgG human antibodies is of broad interest for imaging and treatment of acute coronary syndromes. The objective of our work was to design an optimized production of one selected anti-alphaIIbbeta3-binding scFv fragment for subsequent in vivo animal studies. Fed-batch fermentation was initiated with 2TY media supplemented with 0.1 M glucose. This growing batch culture was used as a starting point for further fed-batch induction, in which a media without glucose containing 1 mM IPTG and 0.4 M saccharose was continuously added. Subsequent purification was performed on the whole cell extract in native conditions over an immobilized copper-ion affinity column. The improved conditions allowed the recovery of 5 mg of highly purified scFv fragments as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The bioactivity of the scFv fragments was further monitored by ELISA, cytometric and immunohistochemical methods.
Robert
R
R
CNRS UMR 5536 Résonance Magnétique des Systèmes Biologiques (RMSB), Université Victor Segalen Bordeaux 2, Bât. 4a, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France.
Noubhani
A M
AM
Jacobin
Marie-Josée
MJ
Santarelli
X
X
Clofent-Sanchez
G
G
D000818:Animals; D001792:Blood Platelets / Q000187:drug effects; D002846:Chromatography, Affinity / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D005285:Fermentation; D005434:Flow Cytometry; D007128:Immunoglobulin Fragments / Q000302:isolation & purification*; D007135:Immunoglobulin Variable Region / Q000302:isolation & purification*; D007150:Immunohistochemistry; D008297:Male; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000276:immunology* / Q000494:pharmacology; D011817:Rabbits; D013927:Thrombosis / Q000276:immunology; D014162:Transfection
D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019039:Platelet Glycoprotein GPIIb-IIIa Complex