Protein Expression in Mammalian Cells Methods and Protocols
Through all of the recent progress provided by high throughput DNA sequencing technologies, it has become clearer and clearer that the study of proteins and protein organelles will be the key to unlocking our ability to manipulate cells and intervene in h
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oduction Recombinant proteins (r-proteins) have many applications in fundamental and clinical research. Until recently, the main technique for obtaining a sufficient amount of an r-protein from mammalian cells was the generation of a stable cell line through a lengthy procedure that included gene delivery, genetic selection, and cell cloning (1). Large-scale transient gene expression (TGE) is being developed as a faster and more economical alternative for producing r-proteins for various research applications (2, 3). With this approach, milligram to gram quantities of a protein can be produced within days after construction of the expression vector. Proteins localized to the nucleus, cytoplasm, or plasma membrane in addition to secreted proteins have been expressed using TGE (4).
James L. Hartley (ed.), Protein Expression in Mammalian Cells: Methods and Protocols, Methods in Molecular Biology, vol. 801, DOI 10.1007/978-1-61779-352-3_2, © Springer Science+Business Media, LLC 2012
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L. Baldi et al.
Recently, volumetric productivity by TGE has passed the 1 g/L barrier at small scale using HEK-293 cells by increasing the cell density at the time of transfection and by adding VPA, a histone deacetylase inhibitor (5–7). TGE is a relatively simple procedure that does not require expensive equipment – only a simple cell culture incubator and an orbital shaker are necessary. Here, we describe the procedure as applied to a one-liter culture in an orbitally shaken 5-L glass bottle, but the same method can be performed in other containers, including Erlenmeyer flasks, spinner flasks, Wave and stirred-tank bioreactors (8, 9). The method is fully scalable and can be performed in either smaller or larger volumes than those described here. The process can be divided into three steps (1) cell culture scale-up to the desired biomass; (2) transfection; and (3) production in a batch or fed-batch mode. To produce a r-protein by large-scale TGE, it is necessary to have (1) a simple assay for its quantification to facilitate the optimization of the transfection and (2) a method for its purification. Here, we describe the production of a human monoclonal antibody from two co-transfected plasmids carrying the full-length cDNAs of the IgG light and heavy chain genes (10, 11). A third plasmid carrying the enhanced green fluorescent protein (eGFP) gene is also co-transfected to have a convenient method (eGFPspecific fluorescence) to measure the efficiency of the transfection. The methods for the purification of the antibody by affinity to protein A and its quantification with an enzyme-linked immunosorbent assay (ELISA) are also described. Although the latter methods are specific for recombinant antibodies, the TGE protocol itself can be applied to any r-protein with slight modifications of some parameters.
2. Materials 2.1. Cell Culture
1. HEK-293 cells adapted to cultivation in serum-free suspension (12). 2. Cylindrical and square-shaped glass bottles with nominal volumes of 100 mL to 5 L (Schott Glass, Mainz, Germany). 3. Ex-cell® 293 medium w
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