Mammalian cells, cloning host

For other production hosts (yeast, insect, and mammalian cells), standard promoter formats have been used in combination with FITP cloning methods to produce vectors for expression screening (see Section 2.3.2). A particularly interesting development is the use of multipromoter plasmids for expression in two or more hosts from a single vector. The construction of a dual E.coli (T7 promoter) and baculovirus transfer vector (polH promoter) for expression in insect cells has been described (Chambers et al., 2004). A three-promoter vector (T7, plO, and hCMV or CAG promoter) is available from Novagen (pTrlEX ) and its use reported for comparing protein expression in E. coli and insect cells (Xu and Jones, 2004). 

Effective cloning systems are available for a variety of bacterial hosts, including Bacillus subtilis, Streptomyces spp., and Agrobacter tumefaciens. Cloning systems have also been developed for eukaryotic hosts such as the yeast Saccharomyces cerevisiae, mammalian cells in tissue culture, and plant cells. 

There exist a variety of vectors for cloning into eukaryotic systems, ranging from yeast (Saccharomyces as well as Pichia) through insect cells (Baculovims) and plants (Ti plasmid from Agrobacterium tumefaciens) to mammalian cells (transfected by viral or mammalian vectors). As expression in eukaryotic hosts is less efficient than bacterial expression in terms of yield and time and more complicated in terms of vector structure and culture conditions, such eukaryotic expression systems are only used for genes whose proteins require posttranslational modification which is not possible in bacteria. Yeast is the preferred option as a relatively easily culturable single-cell system but posttranslational modification capabilities is limited. The additional complexity can be circumvented in part by exploiting the ability of eukaryotic vectors to act as shuttle vectors, which can be shuttled between two evolutionarily different hosts. Thus, eukaryotic vectors can be replicated and analyzed in bacteria and transfected into eukaryotic cells for expression of the recombinant product. 

For the expression of recombinant food allergens, the genomic- or cDNA-derived clone is ligated into a vector and introduced into the host organism. Bacteria, yeast, plants, and mammalian cells can be host systems. 

Second, the flow cytometric isolation of very rare clones from a library is technically demanding. For most protein engineering applications microorganisms are used as the expression host. Whereas the sorting of rare mammalian cells is widely practiced in cell biology research and in clinical medicine and is therefore highly advanced, the screening and isolation of microbial cells is still much more of an art (Davey and Kell, 1996). 

Goto, C., Akai, K., Murakami, A., Hashimoto, C., Tsuda, E., Ueda, M., Kawanishi, G., Takahashi, N., Ishimoto, A., Chiba, H., and Sasaki, R. (1988). Production of recombinant human erythropoietin in mammalian cells host-cell dependency of biological activity of the cloned glycoprotein. Bio/Technology 6, 67 71. 

For higher eukaryotes, the most common approach to cloning is PCR amplification from cDNA libraries in order to avoid the intron problem. Another challenge when expressing eukaryotic proteins is that eukaryotic proteins frequently do not fold properly in E. coli and will not be processed with the correct post-translational modifications, and therefore often other eukaryotic expression hosts must be used. Options include using yeast cells, insect cell lines or mammalian cell lines. 

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