A nanobody-based strategy for rapid and scalable purification of human protein complexes

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 EuB . 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. Key points The protocol describes the lentivirus-based expression of high amounts of soluble and membrane-bound tagged proteins of interest (POI) in human suspension cells. This is combined with rapid nanobody-based purification of the POI and its complexes for downstream structural analysis and functional assays. The protocol provides guidelines for the isolation of a POI in its tagged (TagON) and scarless untagged (TagOFF) forms using an engineered SUMO protease (SENP EuB ). Structural and functional analysis of human proteins requires their isolation in high yields and purity. Here, protease-cleavable, high-affinity, tag-specific nanobodies are used for the isolation of soluble and membrane proteins from lentiviral-transduced human cell suspensions.