A novel epitope tagging system to visualize and monitor antigens in live cells with chromobodies

Bjoern Traenkle,Philipp D Kaiser,Funmilayo O Fagbadebo,Sören Segan,Ulrich Rothbauer

doi:10.1038/s41598-020-71091-x

Bjoern Traenkle, Philipp D Kaiser + Show 3 more

Open Access

https://doi.org/10.1038/s41598-020-71091-x

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Abstract

Epitope tagging is a versatile approach to study different proteins using a well-defined and established methodology. To date, most epitope tags such as myc, HA, V5 and FLAG tags are recognized by antibodies, which limits their use to fixed cells, tissues or protein samples. Here we introduce a broadly applicable tagging strategy utilizing a short peptide tag (PepTag) which is specifically recognized by a nanobody (PepNB). We demonstrated that the PepNB can be easily functionalized for immunoprecipitation or direct immunofluorescence staining of Pep-tagged proteins in vitro. For in cellulo studies we converted the PepNB into a fluorescently labeled Pep-chromobody (PepCB) which is functionally expressed in living cells. The addition of the small PepTag does not interfere with the examined structures in different cellular compartments and its detection with the PepCB enables optical antigen tracing in real time. By employing the phenomenon of antigen-mediated chromobody stabilization (AMCBS) using a turnover-accelerated PepCB we demonstrated that the system is suitable to visualize and quantify changes in Pep-tagged antigen concentration by quantitative live-cell imaging. We expect that this novel tagging strategy offers new opportunities to study the dynamic regulation of proteins, e.g. during cellular signaling, cell differentiation, or upon drug action.

Highlights

Epitope tagging is an effective way to facilitate expression and purification of recombinant proteins and a favored approach to study biogenesis, localization and molecular interactions of proteins of interest (PoIs) when lacking well-defined and reproducible capture and/or detection reagents. In this context we and others have developed nanobodies which on multiple occasions have proven to be valuable and versatile tools for applications ranging from one-step purification to super-resolved imaging of proteins comprising a small peptide t ag[26,27,28,35]
We build on previous work describing a nanobody in complex with a short helical peptide within the gp[41] protein of H IV29 and designed numerous mammalian expression constructs displaying the minimal (12 AA) epitope (PepTag) either on the C- or N-terminus
We demonstrated the capability of functionalized versions of the nanobody (PepNb) for immunoprecipitation (IP) and direct immunofluorescence (IF) of Pep-tagged proteins

Summary

Objectives

Visualizing the PepCB, labeled either with GFP or RFP, we detected in V IMPep expressing cells characteristic filamentous structures in the cytoplasm and for PCNAPep expressing cells typical DNA replication foci within the nucleus (Fig. 3D, E). we aimed to visualize microfilaments and mitochondria using the PepCB

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