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 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.
Wu
Chia-Hung
CH
Institute of Biomedical Sciences, Academia Sinica, Yen Geo Yuan Road, Section 2, No. 128, Taipei, Taiwan.
Balasubramanian
Wesley Roy
WR
Ko
Ya-Ping
YP
Hsu
George
G
Chang
Shih-En
SE
Prijovich
Zeljko M
ZM
Chen
Kai-Chuan
KC
Roffler
Steve R
SR
D000818:Animals; D041702:BALB 3T3 Cells; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D006801:Humans; D051379:Mice; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis*
D011994:Recombinant Proteins