Expression Using Mammalian Cells

Puromycin. Puromycin (19), elaborated by S. alboniger (1—4), inhibits protein synthesis by replacing aminoacyl-tRNA at the A-site of peptidyltransferase (48,49). Photosensitive analogues of (19) have been used to label the A-site proteins of peptidyltransferase and tRNA (30). Compound (19), and its carbocycHc analogue have been used to study the accumulation of glycoprotein-derived free sialooligosaccharides, accumulation of mRNA, methylase activity, enzyme transport, rat embryo development, the acceptor site of human placental 80S ribosomes, and gene expression in mammalian cells (51—60). 

Tanahashi, H., etal. (1990). Photoprotein aequorin use as a reporter enzyme in studying gene expression in mammalian cells. Gene 96 249-255. 

Normal circulating levels of tPA are low, so that to accomplish this dramatic clot breakdown one would need the amount of tPA contained in 50,000 liters of blood. This is clearly not practical. Instead, the molecule has been cloned and expressed in mammalian cells so that it can be produced in quantity. Using cells from mammals, rather than bacteria, results in a product molecule that has the same folding, internal bonding, and coat of sugar residues as the natural protein. 

Witchel, H.J., Milnes, J.T., Mitcheson, J.S., and Hancox, J.C., Troubleshooting problems with in vitro screening of drugs for QT interval prolongation using HERG K+ channels expressed in mammalian cell lines and Xenopus oocytes, J. Pharmacol. Toxicol. Methods, 48, 65-80, 2002. 

The experiment in Figure 13-3 illustrates the use of flow cytometry to determine Kj and B ax values for binding of the es nucleoside transporter ligand, SAENTA-fiuorescein, at sites in human CEM leukemic lymphoblasts. The es nucleoside transporter is widely expressed in mammalian cells, and is concentrated in regions in the CNS that coordinately express adenosine Ai receptors (Jennings et al., 2001). 

Expression by transient transfection methods using mammalian cell lines is a convenient and rapid method of producing recombinant proteins when E. coli systems fail to produce correctly folded, structurally homogeneous protein. Moreover, it is a method that is routinely used to produce proteins for crystallization. 

Other protocols involve cell lines with integrated rep/cap cassettes (Clark et al., 1995 Gao et al., 1998 Liu et al., 1999 Chadeuf et al., 2000 Mathews et al., 2002 Qiao et al., 2002a,b) infected with adenovirus or, alternatively, a recombinant herpesvirus system has been used to provide both helper virus function and rep/cap (Conway et al., 1997, 1999). In a switch away from using mammalian cell and helper virus production systems, rAAV vectors have been made in insect cells where the AAV genes are expressed under the control of insect promoters and the traditional helper virus gene products are not required (Urabe et al., 2002). Stable producer cell... 

Kaufman RJ (1990), Vectors used for expression in mammalian cells, Methods... 

There is a great range of recombinant enzymes used for therapeutic purposes, but not all of them are expressed in mammalian cells. 

Antibodies are expressed by hybridoma cells formed by cell fusion of sensitized animal or human B lymphocytes with myeloma cells, or they are generated by EBV (Epstein-Barr virus) transformation of sensitized B lymphocytes. Other heterologous expression systems such as bacteria, yeast, insect cells, and mammalian cells have also been used for expression of antibodies and their fragments. However, because of renaturation problems, glycosylation, and expression levels, mammalian cells are mostly used for the expression of monoclonal antibodies. More recently, technologies have been extensively developed for the expression of antibodies in transgenic animals and transgenic plants. 

Cheng, L., Fu, J., Tsukamoto, A. and Hawley, R. G. (1996). Use of green fluorescent protein variants to monitor gene transfer and expression in mammalian cells. Nat. Biotechnol. 14,606-609. 

Hong, Z., Ferrari, E., Wright-Minogue, J., Chase, R., Risano, C., Seelig, G., Lee, C.-G., and Kwong, A. D. (1996) Enzymatic characterization of hepatitis C virus NS3/4A complexes expressed in mammalian cells using the herpes simplex virus amplicon system../. Virol. 70,4261—4268. 

The major advantage of using mammalian cells to produce heterologous eukaryotic proteins are that the expressed proteins are correctly folded and glycosylated to near native structures [ 173]. Because of these unique post-trans-lational modifications, various mammalian expression systems have been reported time to time for the production of therapeutic proteins [173-176]. However, the high cost of cell culture, low productivity (5-50 pg cell 1 day-1), and difficulty in growing in large scale imposes serious restrictions on their... 

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