Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Daniel L. Kober; Zeynep Yurtsever; Thomas J. Brett

doi:10.3791/53445

JoVE Journal
Biology

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JoVE Journal Biology

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

高效的哺乳动物细胞表达和细胞外糖蛋白的结晶一步纯化

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07:08 min

December 23, 2015

DOI: 10.3791/53445-v

Daniel L. Kober*^1,2,3, Zeynep Yurtsever*^2,3,4,5, Thomas J. Brett^2,3,5,6,7

¹Molecular Microbiology and Microbial Pathogenesis Program,Washington University School of Medicine, ²Department of Internal Medicine,Washington University School of Medicine, ³Drug Discovery Program in Pulmonary and Critical Care Medicine,Washington University School of Medicine, ⁴Biochemistry Program,Washington University School of Medicine, ⁵Center for the Investigation of Membrane Excitability Diseases,Washington University School of Medicine, ⁶Department of Biochemistry and Molecular Biophysics,Washington University School of Medicine, ⁷Department of Cell Biology and Physiology,Washington University School of Medicine

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这是一种快速、经济高效的方案，用于生产分泌型糖基化哺乳动物蛋白，随后进行单步纯化，获得足够产量的均相蛋白，用于 X 射线晶体学和其他生物物理研究。

这种哺乳动物蛋白质表达技术的总体目标是产生毫克级的天然折叠蛋白质，适用于结构、生物物理和功能研究。该技术的主要优点是它是一种快速直接的方案，用于获得分泌的哺乳动物蛋白和结构研究所需的纯度浓度。一般来说，我们发现蛋白质产量的大多数问题都是由于细胞健康和活力造成的。

通过密切监测细胞活力和培养基糖水平，您可以大大提高蛋白质产量并延长细胞的实用性。监测细胞密度也非常重要。我们发现，如果在转染前的任何时间细胞超过每毫升 200 万个细胞，则蛋白质产量会大大降低要进行 2 93 F 细胞的大规模培养，请补充 1 升 2 93 F 培养基，其中含有 10 毫升 100 x 谷氨酰胺和 5 毫升 100 x 笔链球菌。

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