Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Summary

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

Abstract

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.

BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Introduction

650,000 protein-protein interactions are estimated to exist in the human interactome, playing critical roles in maintaining normal cell functions ^1,2. Besides co-immunoprecipitation (co-IP), the gold standard to study protein-protein interaction from cell lysate, several protein fragment complementation assays (PCA) have been developed to improve sensitivity in detecting protein-protein interaction inside cells ³. Techniques include Förster resonance energy transfer (FRET), Bioluminescence Resonance Energy Transfer (BRET), and Bimolecular Fluorescence Complementation (BiFC) ^4,5. BiFC is based on the facilitated association of two ....

Protocol

1. Cell Transfection

1. Plate cells the day before transfection, plating fewer cells for immunofluorescence.
   1. For immunofluorescence: in a 6-well plate, coat glass cover slips (1 per well) with 0.02% gelatin at 37 °C for at least 4 hr, wash once with medium, and for each well, plate 1 x 10⁵ HEK 293T cells in 2 ml complete growth medium [Dulbecco's modified Eagle's media (DMEM) plus 10% FBS].
   2. For flow cytometric and Western blot analyses: plate 1.2 x 10⁵ HEK 293T cells/well in 2 ml complete growth medium in 6-well plate (0.3 x 10⁵ HEK 293T cells in 0.5 ml complete growth medium per well in 24-well plat....

Results

AKAP-Lbc and PDE4D3 constructs (Figure 1B) were fused to VN and VC fragments, respectively. Interaction of AKAP-Lbc and PDE4D3 results in functional YFP fluorescence (Figure 1A). Here we use the BiFC method to map AKAP-Lbc-binding sites in PDE4D3. Upstream conserved region 1 (UCR1), upstream conserved region 2 (UCR2) and catalytic region (CAT) are conserved among PDE4 family proteins, therefore, full length (FL), UCR1 and UCR2 plus CAT were used for screening the binding site of PDE4D3 t.......

Discussion

BiFC is a simple and sensitive method to study protein-protein interaction. This method cannot be used to identify new protein-protein interactions, however, it is especially convenient to confirm protein-protein interaction inside cells and to study functional properties; such as subcellular localization of protein complexes, mapping of protein-protein interaction sites, and for screening of small molecules/peptides that can modulate protein-protein interaction. Because VN and VC fragments are non-fluorescent, backgroun.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
REAGENTS
Dulbecco’s modified Eagle’s media (DMEM)	Invitrogen	11965-092
Penicilin-Streptomycin (pen/strep), 100x	Invitrogen	15070-063
Fetal Bovine Serum (FBS)	Invitrogen	16000-044
Anti-Flag antibody	Sigma	M2, F1804
Anti-HA antibody	Covance	16B12, MMS-101R
α-tubulin	Sigma	DM1A, T9026
Anti-GFP antibody	Clontech	632569
Cell culture plates, 6-well tissue culture treated	Thermo Fisher Scientific	130184
HEK 293T cells	ATCC	CRL-11268
Maxiprep plasmid purification kit, high speed	Qiagen	12663
Dulbecco’s Phosphate-buffered saline (DPBS), sterile 1x	Invitrogen	14190-144
Trypsin-EDTA, 0.05% (w/v)	Gibco	25300
Polystyrene round-bottom tubes for FACS staining	BD Biosciences	352052
Paraformaldehyde	Fisher Scientific	S74337MF
Prolong gold antifade reagent with DAPI	Invitrogen	P-36931
Superfrost plus Microscope slides	Fisher Scientific	12-550-15
Fisherfinest premium cover glass	Fisher Scientific	12-548-5P
EQUIPMENT
CO2 air-jacketed Incubator	NuAIR DH autoflow
Confocal microscope LSM510 META	Carl Zeiss, Inc
Electrophoresis and transfer unit	Biorad
Cyan ADP Flow Cytometer	Beckman Coulter