DeMaria Christine T
Therapeutic Protein Expression, Genzyme Corporation, Framingham, MA, USA.
Methods Mol Biol. 2012;801:27-39. doi: 10.1007/978-1-61779-352-3_3.
A flow cytometry method using a nonfluorescent reporter protein was developed for rapid, early-stage identification of cells producing high levels of a recombinant protein of interest. A cell surface reporter protein is coexpressed with the protein of interest, and the reporter protein is detected using a fluorescently labeled antibody. The genes encoding the reporter protein and the protein of interest are linked by an IRES so that they are transcribed in the same mRNA but are translated independently. Since they each arise from a common mRNA, the reporter protein's expression level accurately predicts, on a per cell basis, the relative expression level of the protein of interest. This method provides an effective process for selecting cells that express high levels of recombinant proteins, with the benefits of rapid and accurate 96-well plate clone screening (that is both quantitative and qualitative) and elimination of unstable clones during subsequent scale up and culture. Furthermore, because this method does not rely on the availability of a detection reagent specific for the protein of interest that is expressed, it can be easily implemented into any cell line development process.
开发了一种使用非荧光报告蛋白的流式细胞术方法，用于快速、早期鉴定产生高水平目标重组蛋白的细胞。细胞表面报告蛋白与目标蛋白共表达，使用荧光标记抗体检测报告蛋白。编码报告蛋白和目标蛋白的基因通过内部核糖体进入位点（IRES）连接，以便它们在同一mRNA中转录，但独立翻译。由于它们均来自共同的mRNA，报告蛋白的表达水平在每个细胞的基础上准确预测目标蛋白的相对表达水平。该方法提供了一种有效的过程来选择表达高水平重组蛋白的细胞，具有快速准确的96孔板克隆筛选（定量和定性）以及在后续放大培养过程中消除不稳定克隆的优点。此外，由于该方法不依赖于对所表达目标蛋白具有特异性的检测试剂的可用性，它可以很容易地应用于任何细胞系开发过程。
