October 1, 2017 (Vol. 37, No. 17)
Amy Landreman Ph.D. Global Product Manager Promega
Christoper T. Eggers, Ph.D. senior research scientist Promega
Cell-Based, Bioluminescent Protein-Tagging System
Control of protein abundance is one of the master regulators of cellular physiology. Understanding normal changes in cellular protein abundance is key to understanding basic cellular biology and the dysregulation that can underlie many disease states.
In drug discovery, the ability to decrease the abundance of specific proteins is emerging as an attractive alternative to traditional small-molecule strategies. Unlike classic approaches designed to inhibit target protein activity, newer technologies often called proteolysis-targeting chimeras (PROTACs) enable the selective degradation of target proteins. This exciting advancement means taking a second look at small-molecule targeting of proteins that previously were challenging or considered to be “undruggable.”
Common approaches for quantifying cellular protein abundance include mass spectrometry analysis or antibody-based approaches such as ELISA and Western blotting. Epitope-tagging methods, widely used for recombinant protein studies, are appealing because the small tags can easily be fused to a protein of interest and detected with specific antibodies. However, these methods are limited in throughput or sensitivity, require high-quality antibodies, and may only yield semi-quantitative results. Promega recently developed an alternative to classic epitope-tagging approaches that brings the simplicity and sensitivity of bioluminescence to studies on protein abundance.
Bioluminescent Tagging System Derived from Small and Sensitive NanoLuc® Technology
We have used NanoLuc Binary Technology (NanoBiT®)1, a two-part complementation system based on NanoLuc luciferase, to develop a novel peptide tag that enables sensitive bioluminescent quantification of proteins with no antibody requirement. The tag, designated High BiT (HiBiT), is only 11 amino acids in length. HiBiT-tagged proteins are measured using detection reagents containing the peptide’s complementing polypeptide, Large BiT (LgBiT), which binds with high affinity to HiBiT (Kd ~1nM) reconstituting bright, luminescent enzyme activity. In contrast to antibody-based detection methods, the HiBiT tagging system provides a highly quantitative measure of protein abundance using simple add-mix-measure detection with over seven logs of linear dynamic range, making it compatible with both bench-level and high-throughput studies of protein abundance.
Measure Endogenous Protein Expression
HiBiT tagging system is sensitive enough to quantitatively measure proteins down to endogenous levels, even for proteins typically maintained at low levels in the cell. In addition, the small 11aa tag efficiently integrates HiBiT into endogenous loci using a rapid CRISPR/Cas9-mediated genome-editing strategy (Figure 1). Endogenous tagging system described knocks-in HiBiT tag at a precise genomic location using a method that parallels traditional vector-based transient transfection without the cloning requirement.2 When used together, HiBiT and CRISPR/Cas9 can quantitatively study protein abundance under endogenous expression conditions with a simple, quantitative detection workflow.
Figure 1. Endogenous HiBiT tagging and detection protocol. The 11aa tag can efficiently integrate HiBiT into endogenous loci using a rapid, cloning-free CRISPR/Cas9 gene-editing strategy as outlined. One can design and order guide RNA, Cas9, and HiBiT donor DNA from a supplier such as IDT, assemble the RNP complex, deliver the RNP complex to the cell, and detect the genetic edit by luminescence (e.g., GloMax® Discover luminometer).
Quantify Protein Stabilization
Hypoxia-inducible factor-1A (HIF1A) is a transcription factor involved in the response to hypoxic conditions and is thought to play an important role in cancer metabolism. Protein levels are kept low under normal basal conditions by prolyl hydroxylation and VHL-directed ubiquitination. Hypoxic conditions or chemical inhibition of prolyl hydroxylases with compounds such as 1,10-phenanthroline lead to rapid accumulation of HIF1A protein. The accumulation of HIF1A was measured by expressing HIF1A-HiBiT fusion from either CMV- or PGK-driven expression constructs using transient transfection or by insertion of HiBiT into the endogenous HIF1A loci using CRISPR/Cas9 gene editing (Figure 2). Low background and wide dynamic range of HiBiT can detect even the low, basal levels of endogenously expressed HIF1A.
We obtained the expected dose-response increase in HIF1A abundance following increased treatment with 1,10-phenanthroline. We also observed that lower levels of protein expression led to a greater fold response of accumulation, with both expression from the weak PGK promoter and endogenous expression following CRISPR/Cas9 insertion displaying the largest-fold change.
Figure 2. Stabilizing HIF1A-HiBiT by incubation with the hypoxia mimetic 1,10-phenanthroline. HeLa cells were transiently transfected with different amounts of CMV- or PGK-driven expression constructs for HIF1A-HiBiT diluted in carrier DNA. In parallel, HiBiT was tagged to the endogenous locus in HeLa cells using CRISPR/Cas9, and a clone was isolated. Cells were plated in 96-well plates and treated the following day for 4 hours with a titration of 1,10-phenanthroline. Nano-Glo® HiBiT Lytic Reagent was added to all wells and luminescence measured after 10 minutes as in Figure 1. Data were normalized to untreated cells.
Assess Targeted Protein Degradation
Bromodomain-containing protein, BRD4, is one attractive target for cancer therapeutics. Compound dBET1 links BRD4 to cereblon, a component of the E3 ligase complex, to induce its degradation
We have used HiBiT tagging to quantify the degradation of BRD4 following dBET1 treatment by expressing HiBiT-tagged BRD4 in HEK 293 cells, either by transiently transfecting different dilutions of TK- or CMV-expression constructs or by introducing the HiBiT tag at the endogenous locus using CRISPR/Cas9 editing (Figure 3). Following a two-hour incubation with dBET1, BRD4 abundance was significantly reduced in the cells. Interestingly, the CRISPR/Cas9-derived cell pool expressing HiBiT-BRD4 at endogenous levels displayed significantly greater degradation compared to transiently expressed protein, demonstrating how overexpression artifacts can hinder studies of protein degradation. The sensitivity of the HiBiT system enabled us to perform these experiments in a pool of edited cells, as opposed to further generating a clonal population.
Figure 3. Targeted degradation of HiBiT-BRD4 by incubating with compound dBET1. HEK 293 cells were transiently transfected using different amounts of HiBiT-BRD4 expression constructs with either the strong CMV promoter or the weak TK promoter diluted in carrier DNA to keep the DNA amount constant. In parallel, a pool of HEK 293 cells expressing HiBiT-BRD4 from the endogenous locus were generated using CRISPR/Cas9. Cells were plated in 96-well plates and treated the following day for 2 hours with a titration of dBET1 to induce degradation. Nano-Glo HiBiT Lytic Reagent was added to all wells, and luminescence was measured after 10 minutes as in Figure 2. Background was subtracted and luminescence data normalized to untreated cells.
Low expression levels are important in limiting experimental artifacts and maintaining proper stoichiometry with interacting proteins, as evidenced by HIF1A and targeted degradation of BRD4. HiBiT-tagged proteins can be expressed either from transfected expression constructs diluted to appropriate levels or from the endogenous locus by knocking in with CRISPR/Cas9. Sensitivity and bright bioluminescent signal of the HiBiT tagging system enable quantitative detection of cellular protein abundance even at these low levels using a simple detection format. To learn more visit www.promega.com.
1. A.S. Dixon, et al. , “NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells,” ACS Chem. Biol. 11, 400–408 (2016).
2. M.K. Schwinn et al., “CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide,” Manuscript submitted 2017.