A simple method for the production of recombinant proteins from mammalian cells

Expression of recombinant proteins in mammalian cells is useful for obtaining products with normal post-translational modifications. We describe a simple and economical method for the production of milligram levels of proteins in murine fibroblasts. Retroviral or LIPOFECTAMINETM (Gibco Laboratories) transduction was employed to generate stable murinefibroblast producer cells. Confluent cultures of stable fibroblast clones were maintained for up to 1 month in 0.5% serum. Culture medium was collected every 2–3 days and polyhistidine-tagged proteins were purified by ammonium sulphate precipitation and Ni2+-nitrilotriacetic acid affinity chromatography. Highly pure, active, glycosylated recombinant proteins, including human b-glucuronidase, mouse b-glucuronidase, aminopeptidase N (CD13) and a single-chain antibody–enzyme fusion protein, were obtained with yields of 3–6 mg/l of culture medium. Fc-tagged proteins were also produced and purified in a single step by Protein A affinity chromatography with yields of 6–12 mg/l. The techniques described here allow simple and economical production of recombinant mammalian proteins with post-translational modifications.