Univ. Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience, IINS, UMR 5297, F-33000 Bordeaux, France.
Univ. Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience, IINS, Bordeaux, France.
Methods Mol Biol. 2021;2305:83-104. doi: 10.1007/978-1-0716-1406-8_4.
Mammalian protein expression systems are ideally suited for the high-level production of recombinant eukaryotic secreted and membrane proteins for structural biology applications. Here, we present genetic transduction of HEK293-derived cells using lentivirus as a robust and cost-efficient method for the rapid generation of stable expression cell lines. We describe the features of the lentiviral transfer plasmid pHR-CMV-TetO, as well as detailed protocols for production of lentiviral particles, determination of functional lentiviral titer, infection of expression cells, culture and expansion of the resulting stable cell lines, their adaptation to adherent and suspension growth, and constitutive or inducible milligram-scale protein production. The typical lead-time for a full production run is ~3-4 weeks, with an anticipated yield of up to tens of milligrams of protein per liter of expression medium.
哺乳动物蛋白表达系统非常适合高水平生产用于结构生物学应用的重组真核分泌和膜蛋白。在这里，我们使用慢病毒对源自 HEK293 的细胞进行基因转导，这是一种快速生成稳定表达细胞系的强大且具有成本效益的方法。我们描述了慢病毒转移质粒 pHR-CMV-TetO 的特点，以及生产慢病毒颗粒、测定功能性慢病毒滴度、感染表达细胞、培养和扩增由此产生的稳定细胞系、使其适应贴壁和悬浮生长以及组成型或诱导型毫克级蛋白生产的详细方案。完整生产运行的典型前置时间约为 3-4 周，预计表达培养基每升的蛋白产量可达数十毫克。
