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Monitoring Protein Dynamics at Endogenous Levels with a Luminescent Peptide Tag

Part # PS318

Abstract

Christopher Eggers, Brock Binkowski, Kristin Riching, Sarah Mahan, Juliano Alves, Marie Schwinn, Braeden Butler, Danette Daniels, Hicham Zegzouti, Thomas Machleidt, Keith Wood, and Frank Fan
Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711 

The 11- amino-acid peptide, HiBiT, represents an ideal tag for studying the protein dynamics of endogenously expressed proteins. Its small size facilitates rapid knock-in of the tag using ribonucleoprotein complexes of Cas9 and gRNA along with synthesized single-stranded oligonucleotide donor DNA. High-affinity complementation of HiBiT with the 18 kDa LgBiT subunit generates the bright, luminescent NanoBiT™ enzyme, enabling sensitive quantification of HiBiT-tagged proteins over 7 orders of magnitude of linear dynamic range. Changes in protein abundance can be monitored in either a lytic endpoint assay using purified LgBiT protein or in a live-cell kinetic assay by expressing the LgBiT subunit in cells. Interactions of a HiBiT-tagged protein with protein fusions to HaloTag can be measured in live cells using bioluminescence resonance energy transfer (BRET). We apply these assays to the induced degradation by PROTAC compounds of the bromodomain-containing protein, BRD4, and show both a decrease of endogenously expressed HiBiT-BRD4 in cell lysates and live cells and increased protein interactions with the E3 ligase complex and ubiquitin. HiBiT can also be used to measure translocation of proteins to and from the cell surface in a live-cell assay that takes advantage of the membrane impermeability of LgBiT. We show that internalization of endogenously expressed HiBiT-EGFR and HiBiT-β2-AR can be measured in a matter of minutes as a loss in extracellular HiBiT signal. Furthermore, phosphorylation of HiBiT-EGFR upon activation can be monitored in a homogeneous assay by measuring BRET from the HiBiT/LgBiT complex to a fluorescently labeled secondary antibody to monitor binding of an anti-phospho antibody.

Printed in USA.