Structural Immunology Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, 20852, USA.
Structural Immunology Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, 20852, USA.
Protein Expr Purif. 2022 Apr;192:106029. doi: 10.1016/j.pep.2021.106029. Epub 2021 Dec 14.
To generate stable cell lines that express high levels of recombinant genes often requires screening of a large number of transfected cells using ELISA. The most widely used alternative to ELISA screening is to use an intracellularly expressed GFP reporter construct which allows sorting of recombinant gene expression cells based on GFP fluorescence intensity. The disadvantage of cell sorting, however, is that the resulting population will be polyclonal with the danger of instability and overgrowth of low producers. In addition, GFP or its variants can be toxic to host cells at high concentrations, and thus may reduce growth and robustness of high producer cells or even cause them to become apoptotic. We have developed a new mammalian expression system in which a recombinant protein and a fluorescence protein, AcGFP1, are expressed on the same plasmid separated by an internal ribosome entry site (IRES). A signal peptide was incorporated upstream of AcGFP1 so that the fluorescent protein is secreted from cells, preventing cellular toxicity from intracellular accumulation and enabling convenient and accurate measurement of the protein. Expression tests of Ebola viral envelope GP1 and HIV gp120 proteins using this expression system in 293-H cells showed recombinant protein expression levels were closely correlated with AcGFP1 yield. Therefore, AcGFP1 can serve as an accurate reporter for recombinant protein expression and measuring AcGFP1 concentration provides a convenient, product independent and universal way for efficient clone screening.
为了生成表达高水平重组基因的稳定细胞系，通常需要使用 ELISA 对大量转染细胞进行筛选。ELISA 筛选的最广泛替代方法是使用细胞内表达的 GFP 报告构建体，该构建体允许根据 GFP 荧光强度对重组基因表达细胞进行分选。然而，细胞分选的缺点是，所得群体将是多克隆的，存在不稳定性和低产者过度生长的危险。此外，GFP 或其变体在高浓度下可能对宿主细胞有毒，因此可能会降低高产细胞的生长和健壮性，甚至导致它们凋亡。我们开发了一种新的哺乳动物表达系统，其中重组蛋白和荧光蛋白 AcGFP1 在同一质粒上表达，由内部核糖体进入位点 (IRES) 隔开。在 AcGFP1 的上游掺入了信号肽，以便荧光蛋白从细胞中分泌出来，防止细胞内积累造成细胞毒性，并能够方便、准确地测量蛋白质。在 293-H 细胞中使用该表达系统对埃博拉病毒包膜 GP1 和 HIV gp120 蛋白进行的表达测试表明，重组蛋白表达水平与 AcGFP1 产量密切相关。因此，AcGFP1 可以作为重组蛋白表达的准确报告，并且测量 AcGFP1 浓度提供了一种方便、与产品无关且通用的高效克隆筛选方法。
