Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins.,Nature Protocols

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Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins.
Nature Protocols ( IF 13.1 ) Pub Date : 2018-Dec-01 , DOI: 10.1038/s41596-018-0075-9
Jonathan Elegheert _{1,

2,

3} , Ester Behiels _{1,

2,

3} , Benjamin Bishop ₁ , Suzanne Scott _{1,

4} , Rachel E Woolley ₁ , Samuel C Griffiths ₁ , Eamon F X Byrne _{1,

5} , Veronica T Chang ₄ , David I Stuart ₁ , E Yvonne Jones ₁ , Christian Siebold ₁ , A Radu Aricescu _{1,

4}

Affiliation

Structural, biochemical and biophysical studies of eukaryotic soluble and membrane proteins require their production in milligram quantities. Although large-scale protein expression strategies based on transient or stable transfection of mammalian cells are well established, they are associated with high consumable costs, limited transfection efficiency or long and tedious selection of clonal cell lines. Lentiviral transduction is an efficient method for the delivery of transgenes to mammalian cells and unifies the ease of use and speed of transient transfection with the robust expression of stable cell lines. In this protocol, we describe the design and step-by-step application of a lentiviral plasmid suite, termed pHR-CMV-TetO₂, for the constitutive or inducible large-scale production of soluble and membrane proteins in HEK293 cell lines. Optional features include bicistronic co-expression of fluorescent marker proteins for enrichment of co-transduced cells using cell sorting and of biotin ligase for in vivo biotinylation. We demonstrate the efficacy of the method for a set of soluble proteins and for the G-protein-coupled receptor (GPCR) Smoothened (SMO). We further compare this method with baculovirus transduction of mammalian cells (BacMam), using the type-A γ-aminobutyric acid receptor (GABA_AR) β3 homopentamer as a test case. The protocols described here are optimized for simplicity, speed and affordability; lead to a stable polyclonal cell line and milligram-scale amounts of protein in 3-4 weeks; and routinely achieve an approximately three- to tenfold improvement in protein production yield per cell as compared to transient transduction or transfection.

中文翻译：

哺乳动物细胞的慢病毒转导可快速、大规模和高水平地生产可溶性蛋白和膜蛋白。

真核可溶性蛋白和膜蛋白的结构、生物化学和生物物理研究需要以毫克为单位生产。尽管基于哺乳动物细胞瞬时或稳定转染的大规模蛋白质表达策略已得到很好的建立，但它们与高消耗品成本、有限的转染效率或漫长而繁琐的克隆细胞系选择相关。慢病毒转导是将转基因传递至哺乳动物细胞的有效方法，并将瞬时转染的易用性和速度与稳定细胞系的稳健表达结合起来。在本协议中，我们描述了慢病毒质粒套件（称为 pHR-CMV-TetO ₂ ）的设计和逐步应用，用于 HEK293 细胞系中可溶性和膜蛋白的组成型或诱导型大规模生产。可选功能包括荧光标记蛋白的双顺反子共表达，用于使用细胞分选富集共转导细胞，以及用于体内生物素化的生物素连接酶。我们证明了该方法对于一组可溶性蛋白和 G 蛋白偶联受体 (GPCR) Smoothened (SMO) 的有效性。我们使用 A 型 γ-氨基丁酸受体 (GABA _A R) β3 同五聚体作为测试用例，进一步将该方法与哺乳动物细胞的杆状病毒转导 (BacMam) 进行比较。这里描述的协议针对简单性、速度和经济性进行了优化；在 3-4 周内产生稳定的多克隆细胞系和毫克级的蛋白质；与瞬时转导或转染相比，每个细胞的蛋白质产量通常可提高约三至十倍。

更新日期：2019-01-26

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