A Luminescent Pull-Down Approach to Confirm NanoBRET® Protein Interaction Assays

Leta Steffen and Jacqui Méndez-Johnson
Promega Corporation
Publication Date: February 2019; tpub_206

Abstract

Bioluminescent resonance energy transfer (BRET), the transfer of energy from a bioluminescent donor fusion protein to a physically close fluorescent acceptor fusion protein, provides a quantitative method for detecting protein:protein interactions (PPI) in live cells. Although this technique is valuable in understanding PPI and real-time PPI dynamics in live cells, a secondary assay that confirms direct interaction of the two proteins can be useful to confirm the energy transfer results. Copurification pull-down assays can address this need. The NanoBRET® system uses NanoLuc^® luciferase as the energy donor and HaloTag^® protein labeled with the NanoBRET® HaloTag^® 618 fluorophore as the energy acceptor, providing an optimized system for energy transfer experiments. These same tags are also compatible with a streamlined, bioluminescent pull-down method. Here we demonstrate the complete workflow, with additional suggestions for assay controls and optimization.

Introduction

Protein:protein interactions (PPI) are vital for much of cellular function and serve as pharmaceutical targets for human diseases. We developed NanoBRET® technology for improved study of protein interaction dynamics in living cells using HaloTag^® protein and NanoLuc^® luciferase as protein fusion tags. ⁽¹⁾ ⁽²⁾ NanoLuc^® luciferase is a small (19kDa) luminescent reporter engineered to produce a bright, stable, glow-type signal. HaloTag^® protein is a bacterial-derived haloalkane dehalogenase that can be irreversibly linked to a fluorophore through a chloroalkane linker. In a NanoBRET® PPI assay, NanoLuc and HaloTag are fused to the two target proteins of interest and introduced into the cell. HaloTag^® protein is labeled with the fluorescent HaloTag^® NanoBRET® 618 Ligand, which has been optimized as the NanoBRET® energy acceptor. When the two target proteins are in close proximity, energy is transferred from the NanoLuc^® luciferase donor to the HaloTag^® NanoBRET® 618 Ligand acceptor. The NanoBRET® ratio is calculated by dividing the acceptor emission value by the donor emission value, providing a quantitative measure of the interaction between the two target proteins. The bright, blue-shifted signal from the NanoLuc^® donor combined with the far-red-shifted HaloTag^® acceptor create an assay with optimal spectral overlap, as well as increased signal and lower background compared to conventional BRET assays.

When developing a NanoBRET® PPI assay, a secondary approach that confirms the energy transfer results indicate a direct, physical interaction between the two target proteins can be valuable in interpreting results. Copurification pull-down assays are one way to determine whether direct interaction occurs, and the NanoLuc^® and HaloTag^® fusions are well suited to this approach. By interchanging ligands, HaloTag^® protein can be used in a variety of ways, including as a tag on the bait in a pull-down assay. NanoLuc^® luciferase provides a highly quantifiable tag that is easily measured when fused to the prey protein. In a pull-down assay, the HaloTag^® fusion protein binds to HaloLink™ Resin via covalent linkage with a chloroalkane group, making capture of the bait protein more efficient and more sensitive. ⁽³⁾ ⁽⁴⁾ The bait protein and any copurified protein complexes can be eluted using ProTEV Plus protease to cleave the HaloTag^® marker from the fusion protein. The amount of copurified NanoLuc^® fusion protein can then be easily detected by measuring luciferase activity. No additional cloning steps or antibodies are required.

Here we describe a detailed protocol for performing copurification pull-downs using NanoLuc^® and HaloTag^® fusion constructs with two well-characterized PPI assay examples, p53:murine double minute 2 (MDM2) and bromodomain-containing protein 4 (BRD4):histone H3.3 (H3.3). We also provide recommendation for assay optimization and controls.

Methods

Materials:

cultured cells (e.g., HEK293)
cell culture media and reagents
1X PBS, sterile
Nuclease-Free Water (Cat.# P1195)
transfection reagent (e.g. FuGENE^® HD Transfection Reagent, Cat.# E2311)
OptiMEM^® I Reduced Serum Medium (Gibco, Cat.# 31985-062)
NanoBRET® PPI Control Pair (p53, MDM2; Cat.# N1641)
NanoBRET® BRD4/Histone H3.3 Interaction Assay (Cat.# N1830) or other optimized NanoBRET® assay
HaloTag^® Control Vector (Cat.# G6591)
HaloTag^® Mammalian Pull Down System (Cat.# G6504) or HaloTag^® Mammalian Pull-Down and Labeling System (Cat.# G6500)
IGEPAL^® CA-630 (Sigma-Aldrich Cat.# I3021)
ProTEV Plus (Cat.# V6101)
Nano-Glo^® Luciferase Assay System (Cat.# N1110)
NanoBRET® Nano-Glo^® Detection System (Cat.# N1661, N1662, N1663) Includes NanoBRET® Nano-Glo^® Substrate and HaloTag^® NanoBRET® 618 Ligand

Equipment

luminescence reader [e.g., GloMax^® Discover System (Cat.# GM3000)]
refrigerated centrifuge for pelleting cell cultures
2ml Dounce homogenizers with tight pestles OR 25–27-gauge needles
microcentrifuge (preferably refrigerated)
tube rotator

Protein:protein interactions were first measured using optimized NanoBRET® assays in HEK293 cells co-transfected with either p53-HaloTag^®/NanoLuc^®-MDM2 Fusion Vectors or Histone H3.3-HaloTag^®/NanoLuc^®-BRD4 FL Fusion Vectors as described in the NanoBRET® BRD4/Histone H3.3 Interaction Assay Technical Manual #TM444. To confirm protein:protein interactions in pull-down assays (Figure 1), HEK293 cells were plated at 1.44 x 10⁶ cells/6cm culture plate in MEM + 10% FBS + 1X penicillin/streptomycin (n = 3 per test pair) and cultured overnight at 37°C with 5% CO₂. Cells were then transfected with the fusion plasmids and reagent volumes indicated in Table 1 using FuGENE^® HD Transfection Reagent (as described in FuGENE^® HD Transfection Reagent Technical Manual #TM328). In addition to the test pairs, HaloTag^® Control Vector was substituted for HaloTag^® Fusion Vectors with each NanoLuc^® pair as a specificity control. Transfected cells were incubated for a further 24 hours before collection.

Figure 1. Flow diagram of pull-down method.

Table 1. Transfection Conditions for Pull-Down Experiment. HEK293 cells (3.6ml) at 4 × 10⁵ cells/ml were dispensed into 6cm tissue culture plates in triplicate for each of the following conditions. Cells were cultured overnight, then transfected with the reagents and volumes indicated. Before addition, NanoLuc^® fusion plasmids were diluted 1:10 or 1:100 in Nuclease-Free Water, as described in the HaloTag^® Complete Pull-Down System Technical Manual #TM360; the final mass is indicated below. Use the optimized ratio of HaloTag^® and NanoLuc^® fusions for your specific assay as this may vary.

HaloTag^® Fusion Plasmid	HaloTag^® Plasmid (µg)	NanoLuc^® Fusion Plasmid	NanoLuc^® Plasmid (µg)	OptiMEM^® I Reduced Serum Medium (µl)	FuGENE^® HD Reagent (µl)
p53	7.2	MDM2	0.72	200	21
HaloTag^® Control	7.2	MDM2	0.72	200	21
Histone H3.3	7.2	BRD4 FL	0.072	200	21
HaloTag^® Control	7.2	BRD4 FL	0.072	200	21

Cell collection, protein capture and washing were performed as described in the HaloTag^® Mammalian Pull-Down and Labeling Systems Technical Manual #TM342, at reduced scale. In brief, cells were washed, collected in 4ml ice cold PBS, pelleted and frozen at –80°C for at least 30 minutes. Pellets were then thawed, treated with 300µl of Mammalian Lysis Buffer and Protease Inhibitor Cocktail (1X final concentration) on ice for 5 minutes, and homogenized. The lysate was cleared by centrifugation (14,000 × g, 5 minutes, 4°C) and 300µl of cleared lysate was diluted with 700µl of 1X TBS. Diluted lysate was bound to 200µl of pre-equilibrated resin for 15 minutes at room temperature with mixing on a tube rotator. Resin was centrifuged and the pellet washed four times with 1ml of Resin Equilibration/Wash Buffer at room temperature.

Protein complexes were eluted for 1 hour at room temperature with mixing on a tube rotator using 30U (6µl) of ProTEV Plus protease in 50µl of 1X ProTEV Buffer. Tubes were centrifuged (2 minutes, 800 × g) to pellet the resin, and 50µl of supernatant removed to a white 96-well assay plate, along with three wells of 1X ProTEV Buffer for background measurement. The Nano-Glo^® Luciferase Assay System was used to measure the relative amounts of captured prey-NanoLuc^® fusion by adding 100µl of reconstituted Nano-Glo^® Assay Reagent per well. The plate was shaken for 30 seconds, incubated for 3 minutes at room temperature, and luminescence measured on a GloMax^® Discover luminometer. The relative light units (RLU) of background controls were >3 orders of magnitude lower than samples, so background was not subtracted.

Results

NanoBRET® Protein:Protein Interaction Assay

To perform this study, we used two well-characterized protein interactions: p53:MDM2 and histone H3.3:BRD4. Both of these interactions occur constitutively, so no compound treatment was required to induce either interaction.

The transcription factor p53 acts as a tumor suppressor, responding to DNA damage and other cellular stresses to initiate cell cycle checkpoints, apoptosis or both. Under normal conditions, p53 levels are kept low by the ubiquitin ligase, MDM2. The p53:MDM2 interaction was measured using a NanoBRET® Assay in HEK293 cells. We obtained a 4.7-fold increase in NanoBRET® signal compared with no-ligand control (Figure 2). This increase was prevented by overnight treatment with 10µM nutlin-3, a known inhibitor of the p53:MDM2 interaction. Another control using unfused HaloTag^® protein (HT ctrl) instead of the p53-HT further confirms specificity.

Histone H3.3 is a nucleosomal protein commonly associated with transcriptionally active DNA and may also be required for stability of certain heterochromatin structures. BRD4 is a histone acetyltransferase that is involved in transcriptional regulation and epigenetic memory of chromatin structure and is known to interact with histone H3.3. The NanoBRET® Assay was used to measure the interaction of BRD4 and H3.3 in HEK293 cells and showed a 4.6-fold increase in specific NanoBRET® signal compared with no-ligand control between histone H3.3 and BRD4 (Figure 2).

Figure 2. NanoBRET® PPI response in HEK293 cells with the p53:MDM2 and histone H3.3:BRD4 full-length protein pairs. NanoBRET® PPI assays were performed according to the technical manual with each of the protein pairs. Panel A. The p53:MDM2 interaction was studied in the presence of no treatment, 10µM nutlin-3 (Nutlin), or unfused HaloTag^® protein (HT ctrl). Panel B. The NanoBRET® ratio of the histone H2.3:BRD4 interaction. HT = HaloTag; NL = NanoLuc. Mean values ± standard deviation (n = 4) are shown.

HaloTag^® Pull-Down Assay

To confirm the interactions detected by each NanoBRET® assay, pull-down assays were performed using the HaloTag^® Mammalian Pull-Down System. An initial pull-down assay was performed with each protein pair to determine the cell culture scale appropriate (data not shown). Both protein pairs tested generated signals within the linear range of the instrument using input from 6cm plates; other cell types and protein pairs may require higher input (up to 15cm plates) to generate sufficient signal. See Table 2 for suggestions for scaling the transfection protocol.

Table 2. Cell Culture and Transfection Reagents for HaloTag^® Pull-Down Assay. Dilute cells to 4 × 10⁵ cells/ml in appropriate culture medium and plate the indicated volume for the desired assay scale below. Incubate overnight and transfect following the appropriate transfection protocol, using the indicated reagent volumes below. NanoLuc^®-prey DNA should be diluted in Nuclease-Free Water to the ratio optimized for NanoBRET® detection. For example, NanoLuc^®-MDM2 Fusion Vector would be diluted 1:10 and 7.2µl (0.72µg) used for a 6cm plate.

Plate Size	Cells (ml)	HaloTag^®-Bait DNA (µg)	Diluted NanoLuc^®-Prey DNA (µl)	OptiMEM^® I Reduced Serum Medium (µl)	FuGENE^® HD Reagent (µl)
6cm	3.6	7.2	7.2	200	21
10cm	10.0	20	20	600	60
15cm	30.0	30	30	1000	90

Protein pairs were transfected in triplicate for 6cm cultures of HEK293 cells and compared to transfection with the HaloTag^® Control Vector/NanoLuc^® Fusion Vector to assess specificity. Cell lysates were prepared as described above. Protein complexes were captured on HaloLink™ Resin using the HaloTag^® Mammalian Pull-Down System. Bound proteins were eluted using ProTEV Protease, and the copurified NanoLuc^® fusion was measured using the Nano-Glo^® Luciferase Assay System.

Pull-down of MDM2-NanoLuc^® fusion with p53-HaloTag^® bait resulted in 66-fold increase in luminescence compared to a HaloTag^® Control Vector (Figure 3). Pull-down of BRD4-NanoLuc^® fusion with histone H3.3-HaloTag^® bait resulted in a fivefold increase in luminescence compared to a control (Figure 3). These data confirm the NanoBRET® energy transfer results and support the conclusion that there was direct interaction of the protein pairs. Starting from frozen cell pellets, lysis, capture to the resin, elution and detection can be comfortably accomplished in less than 4 hours.

Figure 3. HaloTag^® pull-down from HEK293 cells coexpressing prey-NanoLuc^® fusion and either a HaloTag^®-bait fusion or HaloTag^® control. HEK293 cells were plated in 6cm plates, transfected with the indicated HaloTag^®/NanoLuc^® plasmid pairs or HaloTag^® Control/NanoLuc^® plasmid pairs ("HT Control"), and collected after ~24 hours. Pull-down assays were performed with HaloLink™ Resin and complexes were eluted using 50µl of ProTEV Plus protease. The NanoGlo^® Luciferase Assay was used to measure NanoLuc^® fusion protein in pull-down eluates. Panel A. Pull-down of MDM2-NanoLuc via p53-HaloTag. Panel B. Pull-down of BRD4-NanoLuc via histone H3.3-HaloTag. Mean values ± standard deviation are shown (n = 3).

Pull-Down Assay Optimization

Depending on the protein interaction being studied, optimization of pull-down conditions may be required. Troubleshooting is provided in the HaloTag^® Mammalian Pull-Down and Labeling Systems Technical Manual #TM342 to help guide assay optimization.

Pull-down assays with inducible PPIs may be performed by adding appropriate compounds. We recommend optimizing to identify appropriate treatment conditions and determine if test compounds are required to maintain binding throughout the pull-down assay. Depending on the mechanism of action, you may be required to add test compounds to the lysis and wash buffers.

For DNA-binding proteins, treating with RQ1 RNase-Free DNase (Cat.# M6101) may be required to eliminate false-positive results mediated by DNA binding. Optional treatment with RQ1 RNase-Free DNase is described in the HaloTag^® Mammalian Pull-Down and Labeling Systems Technical Manual #TM342.

Assay sensitivity may be affected by the expression level of the interacting proteins, strength of the interaction, pull-down conditions and the sensitivity of the luminescent plate reader. Some sensitivity issues may be overcome by increasing the scale of the cell culture used. Table 2 includes suggestions for scaling the pull-down assay.

Pull-Down Assay Controls

The data here do not account for differences in transfection and expression efficiency, or binding efficiency with the HaloLink™ Resin, considerations that can be used to further normalize pull-down data. To control for differences in transfection and expression of the NanoLuc^® fusion, reserve 5µl of the diluted lysates on ice. This prebinding control can be diluted with 45µl of 1X ProTEV Buffer and used for measuring total NanoLuc^® Fusion protein expression relative to the eluate using the Nano-Glo^® Luciferase Assay. The luminescence represents 1/200th of the total input.

To measure relative transfection and expression efficiency of the HaloTag^® fusion, reserve an additional 10µl of the diluted prebinding lysate on ice. HaloTag^® fusion partners can also affect the efficiency of covalent capture on the HaloLink™ Resin. To control for capture efficiency, reserve 10µl of the first flowthrough on ice. The HaloTag^® fusion can be measured in the prebinding lysate and flowthrough controls using in-gel fluorescent analysis. In short, HaloTag^® fusion can be covalently labeled with the HaloTag^® TMR Ligand (Cat.# G8251) or HaloTag^® Direct TMR Ligand (Cat.# G2991 or included in Cat.# G6500), electrophoresed on an SDS-PAGE gel, and quantified in the gel using a fluorescent detection scanner (e.g., Typhoon™ FLA 7000 scanner, GE^® Healthcare Life Sciences). See the HaloTag^® Mammalian Pull-Down and Labeling Systems Technical Manual #TM342 for a complete protocol.

Conclusions

The properties of the NanoLuc^® and HaloTag^® proteins make them valuable fusion partners for monitoring protein interaction dynamics in live cells based on energy transfer. These tags are also well-suited for performing pull-down assays that can be used as a secondary approach to confirm protein interaction results obtained in energy transfer assays. By changing the HaloTag^® ligand, you can use a simple purification method and quantify the relative amount of copurified NanoLuc^®-tagged protein in minutes. This provides a streamlined approach that can be performed in less than 4 hours with no additional cloning steps or antibody requirements.

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Article References

Machleidt, T. et al. (2015) NanoBRET—A novel BRET platform for the analysis of protein-protein interactions. ACS Chem. Biol. 10, 1797–804.
Perez-Perri, J.I. et al. (2016) The TIP60 complex is a conserved coactivator of HIF1A. Cell Rep. 16(1), 37–47.
Hook, B. (2014) Cleaner Protein with HaloTag^® Purification Resins Promega Corporation.
Daniels, D.L. et al. (2014) Discovering protein interactions and characterizing protein function using HaloTag technology. J. Vis. Exp. 89, 51553.

How to Cite This Article

Scientific Style and Format, 7th edition, 2006

Steffen, L. and Méndez-Johnson, J. tpub_206 Luminescent pull-down for confirming NanoBRET® PPI. [Internet] February 2019; tpub_206. [cited: year, month, date]. Available from: https://www.promega.com/es-es/resources/pubhub/2019/tpub-206-luminescent-pull-down-for-confirming-nanobret-ppi/

American Medical Association, Manual of Style, 10th edition, 2007

Steffen, L. and Méndez-Johnson, J. tpub_206 Luminescent pull-down for confirming NanoBRET® PPI. Promega Corporation Web site. https://www.promega.com/es-es/resources/pubhub/2019/tpub-206-luminescent-pull-down-for-confirming-nanobret-ppi/ Updated February 2019; tpub_206. Accessed Month Day, Year.