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

نویسندگان

  • Chia-Hung Wu
  • Wesley Roy Balasubramanian
  • Ya-Ping Ko
  • George Hsu
  • Shih-En Chang
  • Zeljko M Prijovich
  • Kai-Chuan Chen
  • Steve R Roffler
چکیده

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 LIPOFECTAMINE (Gibco Laboratories) transduction was employed to generate stable murine-fibroblast 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 Ni(2+)-nitrilotriacetic acid affinity chromatography. Highly pure, active, glycosylated recombinant proteins, including human beta-glucuronidase, mouse beta-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.

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عنوان ژورنال:
  • Biotechnology and applied biochemistry

دوره 40 Pt 2  شماره 

صفحات  -

تاریخ انتشار 2004