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

Stevens, Taylor Anthony and Pinton Tomaleri, Giovani and Hazu, Masami and Wei, Sophia and Nguyen, Vy N. and DeKalb, Charlene and Voorhees, Rebecca M. and Pleiner, Tino (2023) A nanobody-based strategy for rapid and scalable purification of native human protein complexes. . (Unpublished)

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Native isolation of proteins in high yield and purity is a major bottleneck for 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. (1) First, generation of a plasmid coding for a protein of interest fused to an N- or C- terminal GFP or ALFA peptide tag is rapidly achieved using the lentiviral plasmid toolkit we have designed. (2) Human suspension cell lines stably expressing the tagged fusion protein can be generated in <5 days using lentiviral transduction. (3) Leveraging the picomolar affinity of the GFP and ALFA nanobodies for their respective tags, proteins expressed even at low levels can be specifically captured from the resulting cell lysate in a variety of conditions, including detergents and mild denaturants. (4) Finally, rapid and specific elution of tagged or untagged proteins under native conditions is achieved within minutes at 4°C using an engineered SUMO protease. 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; is faster than alternative approaches, requiring ∼8 days from cloning to purification; and results in substantially improved yields and purity.

Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription Paper
Stevens, Taylor Anthony0000-0002-6232-5316
Pinton Tomaleri, Giovani0000-0001-9661-6480
Hazu, Masami0000-0002-6527-9597
Nguyen, Vy N.0000-0002-7563-7386
Voorhees, Rebecca M.0000-0003-1640-2293
Pleiner, Tino0000-0002-5104-0315
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. We thank Pamela Bjorkman for access to her lab’s cell sorter, as well as the Caltech Flow Cytometry facility. This work was supported by: the Heritage Medical Research Institute (RMV), the NIH’s National Institute Of General Medical Sciences DP2GM137412 (RMV), the Deutsche Forschungsgemeinschaft (TP), and the Tianqiao and Chrissy Chen Institute (TP, MH). Data availability statement. The lentiviral transfer plasmids and bacterial expression plasmids described in this study are available from Addgene. Addgene IDs of all plasmids are listed in Table 1. Competing Interest Statement. RMV and GPT are consultants for Gates Biosciences, and RMV is an equity holder.
Group:Heritage Medical Research Institute, Tianqiao and Chrissy Chen Institute for Neuroscience
Funding AgencyGrant Number
Heritage Medical Research InstituteUNSPECIFIED
Deutsche Forschungsgemeinschaft (DFG)UNSPECIFIED
Tianqiao and Chrissy Chen Institute for NeuroscienceUNSPECIFIED
Record Number:CaltechAUTHORS:20230316-181961000.8
Persistent URL:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:120127
Deposited By: George Porter
Deposited On:22 Mar 2023 16:40
Last Modified:22 Mar 2023 16:40

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