Suzuki Jun, Fukuda Machi, Kawata Shigehisa, Maruoka Masahiro, Kubo Yoko, Takeya Tatsuo, Shishido Tomoyuki
Laboratory of Molecular Oncology, Nara Institute of Science and Technology, Ikoma, Nara, Japan.
J Biotechnol. 2006 Dec 1;126(4):463-74. doi: 10.1016/j.jbiotec.2006.04.039.
Conventional stable protein expression systems using mammalian cells include a time-consuming step of antibiotic resistance-based cell cloning. Here, we report a rapid flow cytometry-based method for the collection of retrovirus vector-infected Chinese hamster ovary (CHO) cells that express desired proteins. The vector carries the genes for green fluorescent protein (GFP), as a marker, and glutathione-S-transferase (GST), to express the desired protein as a GST-fusion construct. To render CHO cells susceptible to retrovirus infection, they were forced to express EcoR, the receptor for retroviruses. After infection, cells expressing desired proteins were collected by flow cytometry as a GFP-positive population, and the desired proteins were purified by glutathione affinity chromatography. This method reduces the time required between infection of cells and purification of a desired protein from several months to approximately 2 weeks.
使用哺乳动物细胞的传统稳定蛋白表达系统包括基于抗生素抗性的细胞克隆这一耗时步骤。在此，我们报告一种基于流式细胞术的快速方法，用于收集表达所需蛋白的逆转录病毒载体感染的中国仓鼠卵巢（CHO）细胞。该载体携带绿色荧光蛋白（GFP）基因作为标记，以及谷胱甘肽 - S - 转移酶（GST）基因，以作为GST融合构建体表达所需蛋白。为使CHO细胞易于受到逆转录病毒感染，促使它们表达逆转录病毒的受体EcoR。感染后，通过流式细胞术收集表达所需蛋白的细胞作为GFP阳性群体，并通过谷胱甘肽亲和层析纯化所需蛋白。该方法将细胞感染与所需蛋白纯化之间所需的时间从数月减少至约2周。
