title: Chromatographic purification of the C(H)2 domain of the monoclonal antibody MAK33.
abstract: The C(H)2 domain, one of the constant domains of the murine monoclonal antibody MAK33 (immunoglobulin subtype K/IgG1) was expressed in Escherichia coli forming insoluble inclusion bodies (IBs) and purified by a three-step process including a denaturation-renaturation step, hydrophobic interaction and gel permeation chromatography. After disrupting the cells, the soluble protein fraction was removed by several centrifugation steps. The isolation of the IBs from the cell fragments was achieved by solubilizing the IBs with 6 M guanidinium hydrochloride (GdmCl) and 0.1 M 1,4-dithioerythrit (DTE) to reduce all disulfide bonds. After refolding the C(H)2 domain, 1.5 M (NH4)2SO4 was added to the protein solution in order to precipitate contaminations. Then the protein was loaded on a Butyl-Sepharose fast flow column and eluted with a linear gradient [1.5-0 M (NH4)2SO4]. As the last purification step a gel permeation chromatography was run on a Superdex 75 prep grade. Finally, the purity of the C(H)2 protein was determined by a silver-stained sodium dodecyl sulfate polyacrylamide gel. We achieved a typical yield of 0.5 mg pure protein per 1 g of wet cells.
authors.affiliation: Institut für Organische Chemie und Biochemie, Technische Universität München, Garching, Germany. michael.thies@ch.tum.de
authors.forename: M J
authors.initials: MJ
authors.lastname: Thies
authors.forename: F
authors.initials: F
authors.lastname: Pirkl
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002850:Chromatography, Gel / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D051379:Mice; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013816:Thermodynamics
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins