Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies

The ability to detect and/or manipulate specific cell populations based upon the presence of intracellular protein epitopes would enable many types of studies and applications. Protein binders such as nanobodies (Nbs) can target untagged proteins (antigens) in the intracellular environment. However,...

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Bibliographic Details
Published in:	eLife Vol. 5
Main Authors:	Tang, Jonathan Cy, Drokhlyansky, Eugene, Etemad, Behzad, Rudolph, Stephanie, Guo, Binggege, Wang, Sui, Ellis, Emily G, Li, Jonathan Z, Cepko, Constance L
Format:	Journal Article
Language:	English
Published:	England eLife Science Publications, Ltd 20-05-2016 eLife Sciences Publications Ltd eLife Sciences Publications, Ltd
Subjects:	Animals antibody engineering Antigens Antigens - metabolism Cytological Techniques - methods Developmental Biology and Stem Cells Epitopes Experiments Flow cytometry Gene expression Genetic aspects GFP Health aspects HIV HIV-1 Human immunodeficiency virus Humans Immunoglobulins Intracellular Medical research Medical schools Mice Nanobodies Neuroscience Neurosciences Plasmids Protein Binding Proteins Single-Domain Antibodies - metabolism Software GFP HIV-1 antibody engineering mouse stem cells nanobodies developmental biology neuroscience human
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Summary:	The ability to detect and/or manipulate specific cell populations based upon the presence of intracellular protein epitopes would enable many types of studies and applications. Protein binders such as nanobodies (Nbs) can target untagged proteins (antigens) in the intracellular environment. However, genetically expressed protein binders are stable regardless of antigen expression, complicating their use for applications that require cell-specificity. Here, we created a conditional system in which the stability of an Nb depends upon an antigen of interest. We identified Nb framework mutations that can be used to rapidly create destabilized Nbs. Fusion of destabilized Nbs to various proteins enabled applications in living cells, such as optogenetic control of neural activity in specific cell types in the mouse brain, and detection of HIV-infected human cells by flow cytometry. These approaches are generalizable to other protein binders, and enable the rapid generation of single-polypeptide sensors and effectors active in cells expressing specific intracellular epitopes.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 These authors contributed equally to this work.
ISSN:	2050-084X 2050-084X
DOI:	10.7554/elife.15312