Chromatographic purification of the CH2 domain of the monoclonal antibody MAK33

The CH2 domain, one of the constant domains of the murine monoclonal antibody MAK33 (immunoglobulin subtype κ/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 CH2 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 CH2 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.