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Monitoring intracellular protein interactions using NanoLuc® Binary Technology (NanoBiT™) Brock Binkowski, Christopher Eggers, Braeden Butler, Marie Schwinn, Michael Slater, Thomas Machleidt, Mei Cong, Keith Wood & Frank Fan
Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711-5399
1. Introduction 4. GPCR interaction with β-arrestin-2 7. Agonist-induced RTK dimerization
2. NanoBiT overview 5. RAF dimerization using stable expression 7. Quantifying agonist binding via dimerization
3. Real time kinetics of PPI interaction dynamics 6. Nuclear hormone receptor dimerization 9. Summary
NanoBiT is extremely bright
• Fusion partners can be expressed at very low levels, minimizing potential artifacts
• 100-1,000 fold brighter than split firefly luciferase
NanoBiT components are small & stable
• LgBiT, 18 kDa; SmBiT, 11 amino acids
• LgBiT evolved for increased structural stability, providing a stable fusion partner
NanoBiT is reversible
• Monitor both protein association and dissociation events in real time
NanoBiT offers experimental flexibility
• Monitor protein interaction dynamics at a single time point or continuously for 1-2 hours
• Room temperature or 37 °C measurements
• Validated in 96-, 384- & 1536-well formats
Check http://www.promega.com/nanobit for new NanoBiT PPI expression vectors
For more information on NanoBiT, please see ACS Chemical Biology, 11(2), p. 400-408
www.promega.com 5/2016 Corresponding author: [email protected]
• LgBiT and SmBiT are fused to proteins A & B
• A:B interaction facilitates LgBiT:SmBiT interaction, generating a bright luminescent enzyme
• LgBiT:SmBiT with low affinity (KD = 190 µM), limiting non-specific association and reducing
assay background
• LgBiT:SmBiT interaction is reversible (kon = 500 M-1sec-1; koff = 0.2 sec-1)
• LgBiT evolved for increased structural stability making it a better fusion partner
• Non-lytic assay format allows real-time measurements of protein interaction dynamics for 1-2
hrs
0 2 0 4 0
0
2 5
5 0
7 5
1 0 0
0 .1
1
1 0
1 0 0
1 ,0 0 0
T im e (m in )
Na
no
BiT
, n
orm
Glo
Se
ns
or, n
orm
N a n o B iT G lo S e n s o r
IS O P R O F S K
• Transient expression of SmBiT-PRKACA & LgBiT-PRKAR2A in HEK293
• Modulators of intracellular cAMP added sequentially at indicated time points
• Inverse correlation for NanoBiT vs. GloSensor cAMP 22F (cAMP biosensor)
• NanoBiT can monitor reversible PPIs in real time
0 2 0 4 0
0
5
1 0
1 5
2 0
T im e (m in )
No
rm
ali
ze
d r
es
po
ns
e
A D R B 2 :A R R B 2 A V P R 2 :A R R B 2
2
2
Inactive
PKA
ATP
Agonist
2 1
CA R2A
CA R2A
1 2
R2A
R2A cAMP
1 = SmBiT
2 = LgBiT
1
CA
CA
1
Active
PKA
LgBiT
SmBiT
PM
ARRB2 P
P
PM
ARRB2
Agonist
LgBiT SmBiT
Internalization
Transient or stable association for GPCR:ARRB2
ARRB2 P
P
SmBiT
LgBiT
• Transient expression of ADRB2-LgBiT/SmBiT-
ARRB2 or AVPR2-SmBiT/LgBiT-ARRB2 in
HEK293 cells
• Saturating ISO or AVP added at time zero
• Expected transient interaction seen for
ADRB2:ARRB2 (class A receptor)
• Expected stable interaction seen for
AVPR2:ARRB2 (class B receptor)
• NanoBiT does not interfere with endogenous
biology
+
LgBiT
18 kDa
SmBiT
11 amino
acids
Protein A Protein B A:B interaction
Structural complementation
gives a bright, luminescent
enzyme
• Stable expression of BRAF-LgBiT & CRAF-SmBiT
via bi-directional CMV promoter
• Recombinase-mediated, single copy integration
into HEK293 genome
• Expected rank order potency observed for BRAF
inhibitors that promote dimerization
• Z’ values >0.5 in 384-well format using manual
dispensing
• NanoBiT can be miniaturized to 384- & 1536-
well formats
Protein:protein interactions (PPIs) are essential to the cellular signal transduction pathways
that contribute to cancer. Although numerous approaches exist to monitor PPIs in vitro,
methods for intracellular detection have been more limited. We developed NanoLuc® Binary
Technology (NanoBiT), a two-subunit system based on NanoLuc® luciferase that can be
applied to the intracellular detection of PPIs. Large BiT (LgBiT; 18 kDa) and Small BiT (SmBiT;
11 amino acid peptide) subunits are expressed as fusions to proteins of interest, where PPI
facilitates subunit complementation to give a bright, luminescent enzyme. Unlike related
approaches where an enzyme or protein is simply split, LgBiT was independently optimized for
structural stability and SmBiT was selected from a peptide library specifically for the PPI
application. The result is a subunit pair that weakly associates (KD = 190 µM) yet still maintains
30% of the activity of full-length NanoLuc at saturation. In contrast to many split systems, the
LgBiT:SmBiT interaction is reversible, allowing the detection of rapidly dissociating proteins.
PPI dynamics can be followed in real-time in living cells using the Nano-Glo® Live Cell
Reagent, a non-lytic detection reagent containing the cell-permeable furimazine substrate.
Advantages over split systems include better sensitivity, reversibility, fusion to a peptide or a
small, structurally stable protein domain, real-time measurements using a non-lytic assay
format, and subunits with reduced affinity for self-association.
0 1 0 2 0 3 0
0
2
4
6
8
T im e (m in )
No
rm
ali
ze
d R
es
po
ns
e
H E R 1 :H E R 1
N R G 1E G F V e h ic le
0 1 0 2 0 3 0
0
2
4
6
T im e (m in )
No
rm
ali
ze
d R
es
po
ns
e
H E R 1 :H E R 2
N R G 1E G F V e h ic le
0 1 0 2 0 3 0
0
2
4
6
8
T im e (m in )
No
rm
ali
ze
d R
es
po
ns
e
H E R 1 :H E R 3
N R G 1E G F V e h ic le
0 1 0 2 0 3 0
0
2
4
6
8
T im e (m in )
No
rm
ali
ze
d R
es
po
ns
e
H E R 2 :H E R 3
N R G 1E G F V e h ic le
• The cytoplasmic domain of HER1,
2 & 3 was replaced with linker-BiT
• Transient expression in U2OS cells
• Saturating EGF or NRG1 added at
time zero
• The expected selectivity profile was
seen for EGF and NRG1
• NanoBiT can monitor RTK
homo- or heterodimerization in
real time
HER
1
EGF
HER
1
EGF
0 1 0 2 0 3 0
0
1
2
3
4
T im e (m in )
No
rm
ali
ze
d R
es
po
ns
e
1 E -6
3 .3 3 E -7
1 E -7
3 .3 3 E -8
1 E -8
3 .3 3 E -9
1 E -9
3 .3 3 E -1 0
1 E -1 0
V e h ic le
[E G F ] (g /m l)H E R 1 :H E R 1
-1 1 -1 0 -9 -8 -7 -6 -5
0
1
2
3
4
lo g [E G F ] (g /m l)
No
rm
ali
ze
d R
es
po
ns
e
E C 5 0 = 4 .0 n g /m l
H E R 1 :H E R 1
0 1 0 2 0 3 0
0
2
4
6
T im e (m in )
No
rm
ali
ze
d R
es
po
ns
e
1 E -6
3 .3 3 E -7
1 E -7
3 .3 3 E -8
1 E -8
3 .3 3 E -9
1 E -9
3 .3 3 E -1 0
1 E -1 0
V e h ic le
[N R G 1 ] (g /m l)H E R 2 :H E R 3
-1 1 -1 0 -9 -8 -7 -6 -5
0
2
4
6
lo g [N R G 1 ] (g /m l)
No
rm
ali
ze
d R
es
po
ns
e
E C 5 0 = 9 .3 n g /m l
H E R 2 :H E R 3
• HER truncations used as
described above
• Transient expression in
U2OS cells
• Varying EGF or NRG1
added at time zero
• CRC data at 7 minutes
• Quantify agonist
interaction with RTK via
dimerization
0 2 0 4 0 6 0 8 0
1 0 3
1 0 4
1 0 5
1 0 6
T im e (m in )
Lu
min
es
ce
nc
e (
RL
U)
R 1 8 8 1 (1 0 0 n M )
V e h ic le
R 1 8 8 1
• Transient expression of LgBiT-AR & AR-SmBiT in HEK293 cells
• R1881 agonist added at time zero, which induces AR dimerization
• CRC data plotted at 30 minutes
• Validated for GR homodimerization as well
• NanoBiT can monitor nuclear hormone receptor dimerization in real
time
1 0 0 1 0 1 1 0 2 1 0 3 1 0 4
0
21 0 5
41 0 5
61 0 5
81 0 5
[ In h ib ito r ] (n M )
Lu
min
es
ce
nc
e (
RL
U)
G D C 0 8 7 9A Z 628P L X 4 7 2 0
S o ra fe n ibM L 7 8 6
1 2 3
1 0 3
1 0 4
1 0 5
1 0 6
P la te
Lu
min
es
ce
nc
e (
RL
U)
G D C 0 8 7 9
V e h ic le
Ras Ras
P
B-Raf C-Raf
B-Raf C-Raf
Active
Inhibitor
HER
1
EGF
HER
1
EGF
N D L
N D L
N D L Ag
N D L
-1 2 -1 1 -1 0 -9 -8 -7 -6
0
2 0
4 0
6 0
8 0
lo g [R 1 8 8 1 ] (M )
No
rm
ali
ze
d R
es
po
ns
e