Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA.
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA.
Nat Protoc. 2024 Jan;19(1):127-158. doi: 10.1038/s41596-023-00904-w. Epub 2023 Nov 16.
The isolation of proteins in high yield and purity is a major bottleneck for the analysis of their three-dimensional structure, function and interactome. Here, we present a streamlined workflow for the rapid production of proteins or protein complexes using lentiviral transduction of human suspension cells, combined with highly specific nanobody-mediated purification and proteolytic elution. Application of the method requires prior generation of a plasmid coding for a protein of interest (POI) fused to an N- or C-terminal GFP or ALFA peptide tag using a lentiviral plasmid toolkit we have designed. The plasmid is then used to generate human suspension cell lines stably expressing the tagged fusion protein by lentiviral transduction. By leveraging the picomolar affinity of the GFP and ALFA nanobodies for their respective tags, the POI can be specifically captured from the resulting cell lysate even when expressed at low levels and under a variety of conditions, including detergents and mild denaturants. Finally, rapid and specific elution of the POI (in its tagged or untagged form) under native conditions is achieved within minutes at 4 °C, using the engineered SUMO protease SENP. We demonstrate the wide applicability of the method by purifying multiple challenging soluble and membrane protein complexes to high purity from human cells. Our strategy is also directly compatible with many widely used GFP-expression plasmids, cell lines and transgenic model organisms. Finally, our method is faster than alternative approaches, requiring only 8 d from plasmid to purified protein, and results in substantially improved yields and purity.
高产量和高纯度的蛋白质分离是分析其三维结构、功能和互作组的主要瓶颈。在这里，我们提出了一种使用慢病毒转导人悬浮细胞，结合高度特异性纳米体介导的纯化和蛋白水解洗脱的快速生产蛋白质或蛋白质复合物的简化工作流程。该方法的应用需要预先生成一个编码感兴趣蛋白（POI）的质粒，该质粒融合了 N 端或 C 端 GFP 或 ALFA 肽标签，使用我们设计的慢病毒质粒工具包。然后，该质粒用于通过慢病毒转导生成稳定表达标记融合蛋白的人悬浮细胞系。通过利用 GFP 和 ALFA 纳米体对其各自标签的皮摩尔亲和力，可以从所得细胞裂解物中特异性捕获 POI，即使在低表达水平和多种条件下，包括去污剂和温和变性剂。最后，使用工程化的 SUMO 蛋白酶 SENP，在 4°C 下在 native 条件下快速且特异性地洗脱 POI（标记或未标记形式），只需几分钟即可完成。我们通过从人细胞中高纯度纯化多个具有挑战性的可溶性和膜蛋白复合物来证明该方法的广泛适用性。我们的策略还与许多广泛使用的 GFP 表达质粒、细胞系和转基因模式生物直接兼容。最后，我们的方法比替代方法更快，从质粒到纯化蛋白仅需 8 天，并且产量和纯度显著提高。
