Hopkins Ralph F, Wall Vanessa E, Esposito Dominic
Protein Expression Laboratory, SAIC-Frederick, Inc., Frederick, MD, USA.
Methods Mol Biol. 2012;801:251-68. doi: 10.1007/978-1-61779-352-3_16.
Transient gene expression (TGE) in mammalian cells has become a routine process for expressing recombinant proteins in cell lines such as human embryonic kidney 293 and Chinese hamster ovary cells. The rapidly increasing need for recombinant proteins requires further improvements in TGE technology. While a great deal of focus has been directed toward optimizing the secretion of antibodies and other naturally secreted targets, much less work has been done on ways to improve cytoplasmic expression in mammalian cells. The benefits to protein production in mammalian cells, particularly for eukaryotic proteins, should be very significant - glycosylation and other posttranslational modifications will likely be native or near-native, solubility and protein folding would likely improve overexpression in heterologous hosts, and expression of proteins in their proper intracellular compartments is much more likely to occur. Improvements in this area have been slow, however, due to limited development of the cell culture processes needed for low-cost, higher-throughput expression in mammalian cells, and the relatively low diversity of DNA vectors for protein production in these systems. Here, we describe how the use of recombinational cloning, coupled with improvements in transfection protocols which increase speed and lower cost, can be combined to make mammalian cells much more amenable for routine recombinant protein expression.
哺乳动物细胞中的瞬时基因表达（TGE）已成为在人胚肾293和中国仓鼠卵巢细胞等细胞系中表达重组蛋白的常规方法。对重组蛋白的需求迅速增长，这就需要进一步改进TGE技术。尽管大量的研究集中在优化抗体和其他天然分泌靶点的分泌上，但在改善哺乳动物细胞胞质表达方面所做工作较少。在哺乳动物细胞中进行蛋白质生产的益处，尤其是对于真核蛋白而言，应该是非常显著的——糖基化和其他翻译后修饰可能是天然的或接近天然的，与在异源宿主中的过表达相比，溶解度和蛋白质折叠可能会得到改善，并且蛋白质在其合适的细胞内区室中的表达更有可能发生。然而，由于在哺乳动物细胞中进行低成本、高通量表达所需的细胞培养工艺发展有限，以及这些系统中用于蛋白质生产的DNA载体多样性相对较低，该领域的进展一直很缓慢。在此，我们描述了如何将重组克隆的应用与转染方案的改进相结合，后者可提高速度并降低成本，从而使哺乳动物细胞更适合常规重组蛋白表达。
