Tools for Coproducing Multiple Proteins in Mammalian Cells
Tools for Coproducing Multiple Proteins in Mammalian Cells
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DOI：
10.1007/978-1-61779-352-3_12
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发表时间：
2012-01-01
期刊：
影响因子：
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通讯作者：
Mancia, Filippo
中科院分区：
文献类型：
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作者：
Assur, Zahra;Hendrickson, Wayne A.;Mancia, Filippo
许多哺乳动物系统的结构和功能研究严重依赖于大量的重组多蛋白复合物的供应。哺乳动物细胞即使不是此类实验的唯一合适宿主，也往往是最理想的。这是由于它们产生功能性哺乳动物蛋白质的内在能力。这一优势经常被表达中的产量、产生过表达系所需的时间和升高的成本等问题抵消。多个蛋白质的共表达为这些实验增加了另一个水平的复杂性，因为需要筛选和选择细胞以表达合适水平的每种组分。在这里，我们提出了一个有效的荧光标记程序，建立稳定的细胞系，过表达两种蛋白质的协调，我们验证的方法在生产重组单克隆抗体Fab片段。这一程序可以很容易地扩展到更复杂的系统，包括两个以上的组件。
Structural and functional studies of many mammalian systems are critically dependent on abundant supplies of recombinant multiprotein complexes. Mammalian cells are often the most ideal, if not the only suitable host for such experiments. This is due to their intrinsic capability to generate functional mammalian proteins. This advantage is frequently countered by problems with yields in expression, time required to generate overexpressing lines, and elevated costs. Coexpression of multiple proteins adds another level of complexity to these experiments, as cells need to be screened and selected for expression of suitable levels of each component. Here, we present an efficient fluorescence marking procedure for establishing stable cell lines that overexpress two proteins in coordination, and we validate the method in the production of recombinant monoclonal antibody Fab fragments. This procedure may readily be expanded to systems of greater complexity, comprising more than two components.