Category: Assay Development and Screening
Intracellular signaling is largely mediated through protein dynamics, including changes in protein abundance, interactions, location, or post-translational modification. While the behavior of proteins is commonly studied using overexpressed reporter genes, CRISPR/Cas9 offers the possibility of tagging genes with reporters at the endogenous locus in order to maintain physiological expression levels, regulation, and stoichiometry with binding partners. 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.
Frank Fan– Sr. Director, Research & China Operations, Promega Corp., Madison, Wisconsin
Sr. Director, Research & China Operations
Frank Fan is a Senior Director of Global R&D and China Operations at Promega Corporation. He is responsible for developing novel technologies, assays and products in the area of cellular analysis for the global market and is responsible for operations of Shanghai Promega in China. Frank is a former member of the SLAS Board of Directors and serves as editor on several professional journals with over 30 scientific papers and eight issued or pending patents.
Prior to joining Promega in 2002, Frank was a Senior Investigator in anti-microbial drug discovery at GlaxoSmithKline. Frank pursued his postdoctoral training on microbial molecular genetics in the Department of Molecular Biology and Biophysics at Yale University. He received his Ph.D. in Biochemistry from the University of Iowa, and his Bachelor of Science degree in Biology from NanKai University, Tianjin, China.