4/21/2017. NanoBiT & NanoBRET. New Tools to Study Protein Interaction in Living Cells. Outline. Promega s History of Luciferase-based Systems/Assays

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1 2013 Promega Corporation. Proprietary Information. Not for further distribution. NanoBiT & NanoBRET New Tools to Study Protein Interaction in Living Cells Steffen Barz, PhD Techniocal Service Promega GmbH Outline Luciferases/ Bioluminescent Reporters BRET Assays & Examples of Applications NanoBiT Assays & Examples of Applications Promega s History of Luciferase-based Systems/Assays Reporter Gene Assays GloSensor (camp, Protease Assays) GloResponse (Signaling Pathways) Cell-Health Assays Bioassays (ADCC, PDL1) NanoBRET/NanoBiT (Protein Interaction) Keith V. Wood, Ph.D. 1

2 Introduction Bioluminescent Reporters Advantages of Luciferase-based Assays Photon emission in both cases Primary difference in excitation Advantages ofluciferases asreporterproteins Absence of luciferase activity in most cell types No background high sensitivity Wide dynamic range No post-translational modification is required for enzyme acitivity Non-toxic (even at high concentration) Rapidity (fast detection) Reporter Dynamics NanoLuc Luciferase a new experimental reporter substrate Light substrate Light 130kDa (2x19/2x35 kda) Oplophorus luciferase 7X brighter than Rluc Glow kinetics / broad substrate specificity Shimomura, O., et al. (1978) 19kDacatalytic subunit Light output & stability compromised. Inouye, S., et al. (2000) substrate substrate evolution Hall, M.P., et al. (2012) ACS Chem Biol 7:1848 Furimazine Light Light Light 19kOluc enzyme evolution NanoLuc Luciferase 81,000X NanoLuc Luciferase 2,500,000X Introduction Bioluminescent Reporters Key Characteristics of NanoLuc Luciferase Thermal stable enzyme Active over broad ph range Monomeric enzyme No PTM detected in mammalian cells No disulfide bonds 20 0 Uniform distribution in cells λ (nm) Live Cells Expressing 384- well plate iphone4 Proof of Brightness Relative emission nm 480 nm 565 nm NanoLuc Renilla Firefly 2

3 Introduction Bioluminescent Reporters Applications of NanoLuc Transcriptional Regulation Protein Stability Cell Signaling Endocytosis Compound Screening Biosensors RNA Interference Imaging Protein Interaction Target ID Target Engagement Why Protein Interactions? The study of protein interactions is fundamental for the understanding of cellular processes such as: DNA Replication Transcription Translation Signal-Transduction etc. How are protein interactions studied? Methods for the Identification/ Confirmation/ Study of Protein Interactions Biochemical/ In vitro Methods Co-Immunoprecipitation Protein Pull-Downs Far Western Blotting Mass Spectromertry Cell-Based Methods Two Hybrid Assays FRET BRET Protein Complementation Assays 3

4 Promega s New Tools for the Set-up of Protein Interaction Assays NanoBRET TM NanoBiT TM Receptor Protein-Tag based methods Measurement of protein interactions Real-time measurement in live cells Bioluminescence Resonance Energy Transfer (BRET) Aequorea victoria...in Nature Renilla reniformis Luciferase Donor luciferin oxyluciferin Fluor Acceptor BRET signal Bioluminescence Resonance Energy Transfer (BRET)...in the Lab TRANSFECT FUSION CONSTRUCTS BRET signal X X Y Y ProtX - Luc ProtY - Fluor Luc Donor Luc Donor Conversion of Substrate Conversion of Substrate Fluor Fluor Acceptor Acceptor BRET signal First describedbyy. Xu, et al.proc. Natl. Acad. Sci. U. S. A., 96 (1999), pp

5 How Does BRET Work? Donor = Luciferase Acceptor = Fluorophore Acceptor excitation BRET D A D A Proximity Spectral Overlap Donor Emission Spectral overlap Acceptor Emission BRET Background Acceptor Signal Donor Signal = BRET Ratio Acc sample Don sample Acc donor Don donor = Corrected BRET Ratio Why BRET for Protein-Protein Interactions? Allows real-time measurement in living cells (Mass spec / Pull down / SPR / TR-FRET) Ratiometric assay format insulates against variations in cell number / expression etc (Protein fragment complementation assay) Extremely sensitive towards changes in distance (Pull down/massspec) No physical interaction between donor and acceptor (Protein fragment complementation assay) Donor bleed-through is only source of intrinsic background (FRET) Limitations of Current BRET Systems BRET1 Rluc + coel (480 nm) + YFP (530 nm) Good RET High spectral bleed through Intermediate signal strength / stability Intermediate sensitivity Limited dynamic range Donor BRET Acceptor BRET2 Rluc+ coel hh(400 nm) + GFP2(515 nm) Intermediate RET Little spectral bleed through Poor signal strength / stability BG Limited sensitivity Good dynamic range Excellent Review about BRET Bacart, J, et. al. Biotechnol. J. 2008, 3,

6 Development of NanoBRET TM HaloTag Chloroalkane Fluorescent Molecule Resins Magnetic Resin Glass Slides Modified hydrolase from Rhodocococcus rhodococcus Covalent bond between HaloTag and chloroalkane = irreversible attachment! Workflow NanoBRET TM Assays HaloTag fusion NanoLuc fusion Add compound and fluorescent HaloTag ligand Add NanoLuc substrate Measure emissions & calculate ratio Reading ~450nm and ~610nm emissions 6

7 NanoBRET Optimization: Placement of Donor / Acceptor All possible donor / acceptor pairs should be tested to ensure optimal assay performance GPCR / β-arrestin2 Recruitment Assay + Agonist arr2 NLuc HaloTag arr2 NLuc BRET Argine Vasopressin receptor 2 stimulated with the peptide hormone arginine vasopression (AVP). BRET ratio AVPR2- / NLuc-b-arr AVP [nm] Ready-to-use NanoBRET PPI Assays (~160) Epigenetic protein assays (bromodomain:histone interaction) Signal protein assays (Kras/Braf) Kinase assays (ERK/ELK) Transcription factor assays (cmyc/max) Membrane protein assays (EGFR/GRB2) Please visit our website for a complete list of ready-to-use assays: 7

8 Advantages of NanoBRET Technology Large spectral separation between acceptor and donor Improved dynamic range Bright donor signal enables use of: Low expression levels Intrinsic negative control No separate donor transfection required Promega s New Tools for the Set-up of Protein Interaction Assays NanoBRET TM NanoBiT TM Protein-Tag based methods Measurement of protein interactions Receptor Real-time measurement in live cells Receptor-Ligand-Binding Assays Receptor Tyrosine Kinases GPCRs / 7-TMs P P P P = NanoLuc = Tracer (molecule X coupled to a red fluorophore) 8

9 Screening strategies using NanoBRET Assays based on compound binding to targets in living cells Key Publication: NanoBRET TM ImmunoBRET: Antibody Binding Assay (EGFR) NL NL BRET NL NL Binding kinetics - Cetuximab Binding Specificity Displacement Assay mbret units mbret units BRET units 9

10 2013 Promega Corporation. Proprietary Information. Not for further distribution. Development of a New Structural Complementation Assay NanoBiT TM Promega s New Tools for the Set-up of Protein Interaction Assays NanoBRET TM NanoBiT TM Receptor Protein-Tag based methods Measurement of protein interactions Real-time measurement in live cells Protein Fragment Complementation Assays (PCAs) Protein A Protein B Protein A Protein B Split Reporter Protein Signal Morell M, et. al. FEBS Lett Jun 5;583(11):

11 Protein Fragment Complementation Assays (PCAs) Protein A Protein B Protein A Protein B Split Reporter Protein Signal Disadvantages of currently used complementation assays: Lack of sensitivity strong over-expression required High intrinsic affinity of split reporters self-complementation background Non-reversible only association not dissociation Development of NanoBiT Dissection of NanoLuc NanoLuc topology model: 10x beta barrel Identification of a dissection point Dixon, AS. et al. (2015) ACS ChemBiol Dec 10 Development of NanoBiT Dissected NanoLuc Small BiT Large BiT 11 amino acids 156 amino acids Optimized for low intrinsic affinity Optimized for structural stability 11

12 Development of NanoBiT Optimization of Small BiT Screen peptide library (n = 350) with LgBiT peptides over a 5-log affinity range!! 700 pm 900 nm 190µM Spontaneous complementation Facilitated complementation e.g., protein interactions Key Characteristics of NanoBiT TM Bright Signal of NanoLuc allows low expression levels Small Tag Sizes Low intrinsic affinity of the BiTs (190µM) Reversible: Association & Dissociation Live cell assay High dynamic range Transfection-based NanoBiT TM Workflow 12

13 Cloning of BiT Fusion: Placement of Tags N-Term C-Term Promoter LgBiT/SmBiT Coding Sequence for Protein A ATG TAA N-Term C-Term Promoter Coding Sequence for Protein A LgBiT/SmBiT ATG TAA N-Term C-Term Promoter LgBiT/SmbiT Coding Sequence for Protein B ATG TAA N-Term C-Term Promoter Coding Sequence for Protein B LgBiT/SmBiT ATG TAA Proof of Concept: Protein-Kinase A Model System Proof of Concept: Protein-Kinase A Model System Sequential addition of the following: Isoproterenol (ISO): ADRB2 agonist (camp ) Propranolol (PRO): ADRB2 antagonist (camp ) Forskolin (FSK): activator of adenylyl cyclase (camp ) Result: NanoBiT signal shows good correlation with camp biosensor signal SmBiT-CA:LgBiT-R2A interaction is reversible 13

14 NanoBiT out-performs current state-of-the-art SmBiT - 1 kda NanoBiT LgBiT- 18 kda RLU Fluc(C) -17 kda Split-Firefly Fluc(N) -44 kda Parameter Size Brightness Signal-to-Background Compared to Fluc* 1/3 the size 450X Brighter 50X Higher Dixon, AS. et al. (2015) ACS ChemBiol Dec 10 Annexin-V 35.7 kdaprotein, 4 x 70aa domains Inhibits blood coagulation Binds phosphatidylserine on the outer surface of apoptotic cells in sol n on cells cryoelectron crystalography AV Homomeric latice Van Genderenet al Biochim et BiophysACTA (2008) 1783: Annexin V NanoBiT PPI During Apoptosis LgBiT Anx V Anx V SmBiT CellTox Green CellTox Green Anx V:Anx V interaction Anx V:Anx V interaction Cell membrane Normal cell - Viable TIME PS in inner leaflet Cell membrane intact PS Low AV binding Low NanoBiT interaction Low RLU, Low RFU Apoptotic cell - Viable PS flips to outer leaflet Cell membrane intact High AV binding High NanoBiT interaction High RLU, Low RFU CellTox Green Non-viable cell (2 necrosis) PS in inner & outer leaflet Cell membrane compromised High AV binding High NanoBiT interaction High RLU, High RFU 14

15 2013 Promega Corporation. Proprietary Information. Not for further distribution. NanoBiT TM Annexin V Assay: Real-Time Apoptosis Assays HeLa/ Staurosporin / 8h / 4min acquisition During apoptosis annexin binding increases faster compared to cytotoxicity signal NanoBRET No physical contact for signal generation required Enables analysis of protein interactions with other molecular species Ratiometric (more) complex workflow Specialized instrumentation (filtered luminescence capability) 2 large tags ( / HaloTag) Small dynamic range NanoBiT Simple workflow Large dynamic range Simple luminometer required Small tag size Intensity based assay Limited to protein/protein interactions Signal generation requires physical contact Thank you for your attention! 15