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NanoLuc Complementation Reporter Optimized for Accurate

Measurement of Protein Interactions in Cells
Andrew S. Dixon,†,§ Marie K. Schwinn,† Mary P. Hall,† Kris Zimmerman,† Paul Otto,†
Thomas H. Lubben,† Braeden L. Butler,† Brock F. Binkowski,† Thomas Machleidt,† Thomas A. Kirkland,‡
Monika G. Wood,† Christopher T. Eggers,† Lance P. Encell,*,† and Keith V. Wood†
†
Promega Corporation, Madison, Wisconsin 53711, United States
‡
Promega Biosciences Incorporated, San Luis Obispo, California 93401, United States
*
S Supporting Information
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ABSTRACT: Protein-fragment complementation assays (PCAs) are

widely used for investigating protein interactions. However, the
Downloaded via 83.132.123.215 on September 20, 2019 at 14:49:37 (UTC).

fragments used are structurally compromised and have not been

optimized nor thoroughly characterized for accurately assessing these
interactions. We took advantage of the small size and bright
luminescence of NanoLuc to engineer a new complementation reporter
(NanoBiT). By design, the NanoBiT subunits (i.e., 1.3 kDa peptide, 18
kDa polypeptide) weakly associate so that their assembly into a
luminescent complex is dictated by the interaction characteristics of the
target proteins onto which they are appended. To ascertain their
general suitability for measuring interaction affinities and kinetics, we
determined that their intrinsic affinity (KD = 190 μM) and association
constants (kon = 500 M−1 s−1, koff = 0.2 s−1) are outside of the ranges typical for protein interactions. The accuracy of NanoBiT
was verified under defined biochemical conditions using the previously characterized interaction between SME-1 β-lactamase and
a set of inhibitor binding proteins. In cells, NanoBiT fusions to FRB/FKBP produced luminescence consistent with the linear
characteristics of NanoLuc. Response dynamics, evaluated using both protein kinase A and β-arrestin-2, were rapid, reversible,
and robust to temperature (21−37 °C). Finally, NanoBiT provided a means to measure pharmacology of kinase inhibitors
known to induce the interaction between BRAF and CRAF. Our results demonstrate that the intrinsic properties of NanoBiT
allow accurate representation of protein interactions and that the reporter responds reliably and dynamically in cells.

P roteins are central to the physiological dynamics of living

cells, forming interactive networks that are responsive to
evolving circumstances (e.g., environmental conditions, stress or
pathological situations, or the action of drugs).1 The interactions
arise through a complex interplay of structural conformations,
relationships with other molecules, and microenvironmental
surroundings. Due to the complexity of these functionally
contingent ensembles, analytical methods are needed that do not
disturb the native cellular context. Split reporters have been
commonly used for this purpose, where complementary reporter
fragments are genetically fused onto a pair of interacting
proteins.2 The fragments normally exhibit little reporter activity
separately, but regain activity when brought together through the
interaction of their fused partners.
Although conceptually straightforward, the ability of split
reporters to provide quantitative data without perturbing the
underlying interaction dynamics is not assured. Because
association of the reporter fragments is required for the
reconstituted activity, the intrinsic binding affinity of the
fragments could bias the behavior of their fusion partners. Split
fluorescent proteins, for example, do not reversibly associate, and
chromophore maturation tends to be slow, thus obscuring the
dynamics of the target proteins under study.3 β-Galactosidase
(116 kDa monomer) has also been used as a split reporter,4 but
its size and requirement for oligomerization further impose
interaction relationships that may be inconsistent with the target
proteins. Furthermore, reporters such as β-galactosidase that rely
on the accumulation of an enzymatic product for detection are
hindered in their ability to reveal process dynamics.
Assays based on split luciferases are often preferred due to
their sensitivity and simplicity. Luminescence provides an
instantaneous indication of reporter activity in living cells and
is readily generated by adding substrate to the growth medium.
However, although commonly regarded for producing quanti-
tative data, split luciferases have been evaluated largely for assay
precision rather than accuracy.5−8 While reproducibility and
correlation with known behaviors of interacting proteins have
been shown, quantitative concordance of assay data with the
underlying molecular interaction has been difficult to substan-
tiate. Hence, reinforced by the known limitations associated with
many split reporters, doubts persist on the general suitability of
Received: September 19, 2015
Accepted: November 16, 2015
Published: November 16, 2015

© 2015 American Chemical Society 400 DOI: 10.1021/acschembio.5b00753

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Figure 1. Nluc dissection and optimization. (a) Nluc topology model9 consisting of a 10-stranded beta-barrel. The identified dissection point (red
arrow) occurs between residues 156 and 157 and generates a 156-amino acid polypeptide (156) and a 13-amino acid peptide (native peptide, NP). (b)
Nluc Western blot of HEK293T lysates expressing empty vector (−), 156, 11S, or Nluc. β-actin was included as a loading control. (c) Intracellular half-
life of Nluc, 156, and 11S in HeLa cells following treatment with cycloheximide, as measured by enzyme activity, i.e., normalized relative luminescence
units (RLU; with saturating NP for 156 and 11S). (d) Thermal stability of purified Nluc, 156, and 11S, based on remaining activity at ambient
temperature in the presence of saturating NP added 30 min after temperature challenge. (e) Specific activities of 156 and 11S normalized to Nluc.
Equimolar amounts of each were used to determine Vmax values (under conditions of saturating NP for 156 and 11S). (f) Titration of 11S with various
peptides demonstrating the broad range (>105) of binding affinity. KD values (shown in parentheses) were determined by fitting the data to a one site
specific binding model. For panels c−f: n = 3, variability displayed as SD.

this approach for providing accurate results. Although qualitative
correlation may be sufficient for certain experimental purposes,
accurate representations of intracellular dynamics would
normally be preferred.
NanoLuc (Nluc) is an engineered luciferase derived from a
deep sea luminous shrimp.9 The enzyme is small (19 kDa),
stable, and produces bright and sustained luminescence. Taking
advantage of these attributes, we sought to create a binary
reporter system having minimal steric burden on the fusion
partners but still providing high detection sensitivity. Our
objective was to allow accurate quantitation of protein
interactions under physiological conditions relevant to the
cellular environment. Therefore, in addition to having a minimal
physical presence, our reporter system was designed to have
minimal influence on the affinity and association kinetics of the
interacting target proteins. Furthermore, the reporter was
401
designed to be quantifiable within living cells at relevant
concentrations and temperatures.
We accomplished this through the application of two unique
design strategies. First, beyond simply identifying an optimal split
site, we further enhanced efficacy through structural optimization
of the individual components. Although this approach had
already been applied in the development of Nluc,9 we expanded
it to address issues specific to a dynamic binary structure. The
second strategy resulted from our fortuitous discovery that Nluc
could be dissected 13 amino acids from its C-terminus. We
envisioned that such a small tag would minimize steric conflicts
on fusion partners where geometric constraints may be critical.
This design strategy also allowed reporter stability and intrinsic
affinity to be separately engineered. Stability was optimized in the
larger component, which has sufficient size to retain an
independent tertiary structure. Intrinsic affinity was then
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Figure 2. Influence of NanoBiT on the SME1/BLIP interaction. (a) Inhibition profiles of the SME1/BLIP WT interaction with differing combinations
of fused NanoBiT. Each profile was normalized by the β-lactamase (SME1) activity measured in the absence of BLIP. (b) Inhibition constants (Ki)
determined by β-lactamase activity with different combinations of fused NanoBiT. Corresponding dissociation constants (KD) determined by luciferase
activity where both SME1 and BLIP were fused to NanoBiT. (c) Relationship between NanoBiT luminescence and inhibition of SME1 (both
normalized). (d) Increase in luminescence with time after the addition of BLIP-114 at various concentrations (0.5−30 nM). (e) Observed rate constants
(kobs) for each concentration of BLIP-114. (f) Association (kon) and dissociation (koff) rate constants of BLIP binding to SME1 calculated from NanoBiT
measurements. KD values calculated from the ratio of koff/kon. For all panels: n = 3, variability displayed as SD.

separately adjusted with the smaller component, which is too Our results reveal that simply splitting a reporter protein may
short to support a tertiary structure on its own. not necessarily provide optimal fragments for quantifying protein
402 DOI: 10.1021/acschembio.5b00753
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interactions. Rather, compromised structural integrity resulting
from protein fragmentation can reduce the activity of the
reporter and as a result decrease detection sensitivity. Addition-
ally, the intrinsic affinity of the fragments can limit the dynamic
range of the reporter response and potentially interfere with the
interaction characteristics of the target proteins. By adapting the
structure of these fragments to serve as components of a binary
reporter system, we have improved their analytical proficiency
for revealing dynamic intracellular processes occurring under
physiological conditions. In experiments designed to evaluate
quantitative concordance of the luminescent signal with protein
interactions, this complementation reporter based on Nluc
delivered accurate representations of both affinity and
association kinetics. As the components of this optimized
reporter are structurally distinct from Nluc and for nomenclature
simplicity, we refer to the reporter as NanoBiT (shorthand for
NanoLuc Binary Technology).
■
RESULTS AND DISCUSSION
Engineering a Complementation Reporter. To identify
potential dissection points in Nluc, we created circularly
permutated10 variants in which the N- and C-termini were
relocated within the protein structure, and the original termini
were tethered by a cleavable peptide linker (i.e., containing a
recognition sequence for TEV protease). Ninety variants,
representing permutation sites distributed throughout the Nluc
sequence, were generated and screened in cell lysates for
luciferase activity. Measurements were made both before and
after proteolytic cleavage to identify fragments that require a
proximity constraint for maintaining luciferase activity (Figure
s1a). By this method, a dissection point was found between
residues 156 and 157 (Nluc CP-157; Figure 1a; Figure s1b−g),
leading to an 18 kDa polypeptide fragment (termed 156) and a
much smaller 13-amino acid (1.5 kDa) peptide (termed native
peptide, or NP). The binding affinity (KD = 6 μM) between 156
and NP is comparable to that between fragments of a commonly
used split firefly luciferase (Fluc;11 Figure s2), suggesting their
potential suitability as a complementation reporter. However,
156 expressed poorly in cells (Figure 1b) and was rapidly
degraded compared to full-length Nluc (Figure 1c). This was
likely a result of compromised structural stability associated with
fragmentation, as evidenced by reduced thermal stability of the
luminescent activity (Figure 1d) and formation of insoluble
aggregates in E. coli (Figure s3a).
To improve stability, 156 was randomly mutated and screened
for luminescence in E. coli lysates containing NP. Higher
luminescence should result from increased enzyme expression or
increased specific activity, either of which were expected to
correlate with improvements in structural stability. Two rounds
of mutagenesis and screening (nearly 15,000 variants) uncovered
16 beneficial amino acid substitutions which were combined to
produce the variant, 11S (Figures s3, s4a; Table s1). In
mammalian cells, 11S exhibited greater protein expression
(Figure 1b) and reduced protein turnover (Figure 1c) to levels
comparable with Nluc. Luminescence expression, assayed in the
presence of NP, was also increased by over 300-fold (Figure s4b),
with specific activity increased 3-fold (Figure 1e) to 37% of Nluc.
Thermal stability of the luminescence (i.e., Tm) increased from
48 to 54 °C (Figure 1d).
While appending an unstable polypeptide could potentially
promote degradation of a target protein, it could also result in
suppression of degradation. To investigate this, 11S and 156 were
fused to Fluc-PEST (FlucP),9,12 and the loss of FlucP activity
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following inhibition of protein synthesis was monitored (Figure
s5a,b). No significant effect on protein turnover was evident with
either 11S or parental 156. A slight increase in degradation may
be associated with attaching the unstructured NP (or a modified
variant; Figure s5c−e).
The apparent KD of 11S with NP was 900 nM (Figure 1f;
Table s2), indicating that the affinity may be too high for use with
weakly interacting proteins. Ideally, the intrinsic affinity of the
complementation reporter should be substantially lower than the
affinity of the interacting target proteins (with affinities in the
high nM to μM range). To reduce the intrinsic affinity of the
reporter, a semirandom library of 350 variants of NP comprising
generally conservative amino acid substitutions (or deletions at
each terminus) was synthesized and screened for affinity and
luminescence with 11S. This uncovered complementing
peptides with affinities that spannned 5 orders of magnitude,
yet for many of the peptides the specific activity (i.e., Vmax) across
the variants was relatively unaffected (Figure 1f; Table s2). The
lowest affinity was achieved with 114, an 11-amino acid peptide
(1.3 kDa) exhibiting an apparent KD of 190 μM. The
luminescence spectrum of 11S with 114 is essentially unchanged
from that of Nluc (peak emission ∼460 nm; Figure s6). The
weak association between 11S and 114 indicates that they should
not significantly alter the binding affinity of target proteins having
KD values up to ∼10 μM.
Further investigation revealed that peptides even shorter than
11 amino acids could deliver productive complementation with
11S, although with some loss of light intensity (Table s3; Figure
s7). In addition, we found that a much shorter linker (Table s4;
Figure s8) or no linker at all (Table s5; Figure s9) may be
sufficient for the peptide. Having a shorter complementary
peptide or linker may be beneficial in circumstances where a
minimal fusion tag is preferred.
Accuracy of Interaction Measurements. We used the
interaction between purified SME-1 β-lactamase (SME1) and β-
lactamase inhibitory protein (BLIP)13 as a model to assess the
accuracy of 11S combined with 114 (NanoBiT) for quantifying
protein interactions. This model allowed binding interactions to
be measured either through inhibition of β-lactamase or by
NanoBiT luminescence. Moreover, using reported affinity
mutants of BLIP,14 we could evaluate a range of binding
interactions without altering the overall molecular geometry.
Specifically, we used BLIP WT (Ki = 2.4 nM), BLIP Y50A (Ki =
32 nM), and BLIP R160A (Ki = 322 nM) to provide a ∼100-fold
range in binding affinity.
Evaluation of different linkage orientations revealed optimal
performance with 11S appended to the C-terminus of SME1 and
114 appended to the C-terminus of BLIP. This configuration
apparently imposed minimal steric constraints, since appending
differing permutations of this configuration had little effect on
the inhibition profile of SME1 with the various BLIPs (Figure
2a,b; Figure s10a,b). The estimated Ki values corresponded to
published measurements,14 with small differences between each
other (2-fold or less) associated mostly with fusions of 114 to the
BLIPs. From the configuration containing both subunits of
NanoBiT, the measurements of luminescence corresponded
closely with the inhibition of β-lactamase activity (Figure 2c;
Figure s10c,d). Values for Ki (β-lactamase inhibition) and KD
(NanoBiT bioluminescence) were essentially indistinguishable
within the precision of the assays (Figure 2b), indicating that
NanoBiT provided an accurate assessment of protein inter-
actions in this model system.
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To assess the ability to quantify interaction kinetics, we also
used NanoBiT to estimate the association (kon) and dissociation
(koff) rate constants for SME1 with the BLIPs. In contrast to the
assay for β-lactamase (colorimetric conversion of nitrocefin), the
instantaneous production of luminescence allows dynamic
processes to be continuously monitored. The increase in
luminescence upon combining BLIP with SME1 conformed
well to a pseudo-first-order reaction (Figure 2d; Figure s11a,b),
and kobs values correlated linearly with BLIP concentrations
(Figure 2e; Figure s10c,d). The data indicate that reduced affinity
of the BLIP mutants was achieved largely by increased koff, with
relatively little effect on kon. Values of KD calculated by the ratio of
koff to kon corresponded well to the estimates of KD made under
steady-state conditions (Figure 2f).
The intrinsic kon and koff rate constants for NanoBiT were
determined to be ∼500 M−1 s−1 and 0.2 s−1, respectively (Figure
s12). The BLIP having the lowest affinity (R160A, measured KD
∼ 1 μM) also had the lowest kon and highest koff values. As the kon
for NanoBiT was ∼10-fold lower than for R160A and the koff ∼
10-fold higher, the intrinsic kinetics of NanoBiT should not affect
the interaction kinetics of R160A with SME1. The effect of
NanoBiT should be even less for the higher affinity BLIPs.
NanoBiT may begin to influence the apparent kinetics of protein
interactions having unusually slow association rates or fast
dissociation rates, which may underly very weak binding affinities
(e.g., KD > 10 μM).
Protein Interactions in Cells. For NanoBiT to produce
quantitative data in living cells, its luminescence must be linear
with the intracellular concentration of the binary complex.
Linearity of Nluc luminescence with reporter concentration has
previously been established biochemically.9 In both live cells and
cell lysates, luminescence was also proportional to the amount of
Nluc DNA used for transfection (Figure 3), although deviating
Figure 3. Linearity of luminescence in cells and cell lysates. HEK293T
cells transfected with NanoBiT (equal amounts of FRB-11S and FKBP-
114) or Nluc DNA were treated (±) with 30 nM rapamycin (R) and
measured for luminescence before or after cell lysis. n = 3 (NanoBiT) or
n = 6 (Nluc), variability displayed as SD. Curve fits for Nluc and
NanoBiT were generated based on the expected first order and second
order relationships, respectively.
slightly at high expression. To assess linearity for NanoBiT, the
rapamycin-inducible FRB/FKBP model was used (i.e, FRB-11S/
FKBP-114; Figure s13a,b). At higher DNA concentrations, the
intracellular luminescence from NanoBiT in the presence of
rapamycin followed a linear trend similar to Nluc. The signal
departed significantly at lower concentrations, suggesting some
dissociation of FRB/FKBP even in the presence of rapamycin.
These data conform to a second order model for the
concentration-dependent interaction of two proteins. Lumines-
cence was greatly reduced in the absence of rapamycin (i.e., >
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99%), consistent with essentially complete dissociation of FRB/
FKBP. As expected, the interaction profile in cell lysates shifts
strongly to the right due to dilution of interacting proteins into
the culture medium. The lower baseline results from reduced
spontaneous luminescence (i.e., autoluminescence) generated by
furimazine in the lytic reagent.
Intracellular Kinetics. As cellular processes are generally
reactive to environmental conditions, it is advantageous for the
reporter to also indicate the dynamic character of protein
interactions. This is exemplified by the transent interaction of β-
arrestin-2 (ARRB2) with the β2-adrenergic receptor (ADRB2),
in contrast to the more stable interaction it makes with the
arginine vasopressin receptor 2 (AVPR2).15 This kinetic
distinction was apparent by expressing ADRB2-11S/114-
ARRB2 or AVPR2-114/11S-ARRB2 in HEK293T cells treated
with the appropriate agonist (isoproterenol and vasopressin,
respectively; Figure 4a). Temporal differences were also
observed by bioluminescence imaging of HeLa cells expressing
the NanoBiT fusions (Figure s14a−d).
More kinetically challenging is the interaction of protein
kinase A catalytic (PRKACA) and type 2A regulatory
(PRKAR2A) subunits in response to intracellular cAMP
concentrations. Previous studies have shown that dynamic
changes in the association of these subunits can occur rapidly in
response to applying external stimuli to the cells.15 We explored
the ability of NanoBiT to represent these rapid dynamics by
expressing 114-PRKACA and 11S-PRKAR2A in HEK293T cells,
along with a luminescence-based biosensor for intracellular
cAMP.16 By utilizing a different substrate (Fluc luciferin), the
biosensor can be independently assayed within cells. Modulation
of the endogenous ADRB2 receptor through progressive
addition of an agonist (isoproterenol) and antagonist (propra-
nolol), followed by stimulation of adenylate cyclase with
forskolin, caused rapid changes in cAMP concentrations as
indicated by the biosensor. Corresponding inverse changes were
evident in NanoBiT luminescence, showing precise temporal
coupling of the reporters in response to the underlying cellular
dynamics (Figure 4b).
As molecular kinetics are fundamentally linked to temperature,
measurements at the relevant physiological temperature are
required to accurately represent interaction dynamics. The
response profile of protein kinase A represented by NanoBiT
showed that both association and dissociation rates were much
faster at 37 °C than 21 °C (i.e., ambient temperature; Figure
5a,b). The analogous experiment using split Fluc as the
complementation reporter (i.e., PRKACA-FlucC/FlucN-
PRKAR2A) showed a marked deterioration of the data quality
at physiological temperature. This is consistent with a previous
report describing the unstable nature of split Fluc.17 Because the
114 peptide is unlikely to have conformationally defined
structure in the absence of 11S, the suitable performance of
NanoBiT at higher temperature was likely due to the thermal
stability of the 11S subunit.
Pharmacological Modulation of Protein Interactions.
Because complementation reporters are frequently used to
investigate the influence of synthetic molecules on protein
interactions, we evaluated the suitability of NanoBiT for this type
of application. The effect of intrinsic affinity between the
complementation subunits was investigated by expressing FRB-
11S in HEK293T cells, together with FKBP fused to peptides
with differing affinities to 11S. Our results indicate that the
apparent potency of rapamycin to induce protein dimerization
was minimally affected by the intrinsic affinity (range >100,000
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Figure 4. Monitoring temporal interactions in cells. (a) ARRB2 interaction with fast or slowly recycling GPCRs. The interactions of ADRB2-11S/114-
ARRB2 and AVPR2-114/11S-ARRB2 were followed continuously in HEK293T cells at 37 °C after treatment at t = 1 min with isoproterenol (10 μM) or
Arg8-vasopressin (100 nM), respectively. Individual S/B values were scaled by their respective dynamic ranges by subtracting the minimum value of the
data set, then dividing by the maximum value. (b) Reversible interaction of 114-PRKACA/11S-PRKR2A followed continuously in HEK293T cells at 28
°C. 10 μM isoproterenol (Iso), propranolol (Pro), and forskolin (Fsk) were added sequentially to modulate endogenous ADRB2 (Iso and Pro) or
activate endogenous adenylate cyclase (Fsk). Changes in intracellular cAMP concentration were monitored independently using GloSensor cAMP.
Data were scaled by respective dynamic ranges as in panel a. For both panels: n = 3, variability displayed as SD.

Figure 5. Influence of temperature on monitoring PKA. Comparison of NanoBiT and split Fluc for monitoring PKA interaction dynamics at 21 °C (a)
and 37 °C (b). HEK293T cells transiently expressing PRKACA and PRKAR2A fused to NanoBiT (50 pg DNA/well) or split Fluc (5 ng DNA/well)
were treated as described in Figure 4b. Data were normalized as described for Figure 4b. For both panels: n = 3, variability displayed as SD. Data for other
DNA concentrations can be found in Figure s15.

examined; Figure 6a; Figure s16a−i), and the values achieved are
consistent with published reports.18 This should be expected
since compound potency is typically associated with binding
affinity to its target, rather than interaction affinity within the
target complex. Thus, potency should be minimally influenced by
the choice of complementation reporter so long as the binding
site for the compound is unaltered. In contrast, the dynamic
response induced by rapamycin is strongly dependent on the
intrinsic affinity of the reporter (Figure 6b). This arises primarily
from increased luminescence of the sample lacking rapamycin,
presumably due to increased spontaneous interaction promoted
by the interaction affinity of the reporter subunits. Thus, the
highest assay sensitivity was achieved with the low affinity 114
peptide (S/B = 1,200; Z′-factor =0.98). A similar analysis was
performed with split Fluc (Figure s17).
More pharmaceutically relevant is the interaction of BRAF
with CRAF, which paradoxically is induced by inhibitors
designed for oncogenic mutants of BRAF. Although intended
to promote tumor regression by suppressing the RAS-RAF-
MEK-ERK signaling pathway, these inhibitors can activate the
405
pathway in cells expressing wild type BRAF.19 This phenomenon
has been monitored in cells previously using kinase domains
modified with the CAAX membrane targeting motif.20 We
surmised that the small size and stability of NanoBiT may allow
drug-induced dimerization to be detected in the full-length
proteins. In cells expressing BRAF-11S/CRAF-114, an inhibitor-
dependent increase in dimerization was observed, with the
calculated EC50 for each inhibitor in agreement with previously
described potencies20 (Figure 7a). Cells expressing the NanoBiT
fusions exhibited increased phosphorylation of MEK and ERK in
correlation with drug-induced dimerization21 (Figure s18a,b).
NanoBiT was also capable of differentiating between the
reversible inhibitor, GDC 0879, and the irreversible inhibitor, AZ
628. Removal of GDC 0879 from the cells caused the
luminescence to decrease, indicating dissociation of BRAF/
CRAF. In contrast, removal of AZ 628 had no effect, indicating
that it remained bound to its target (Figure 7b). These results
indicate that the intrinsic affinity of the NanoBiT subunits is
suitable for quantification of both the potency and kinetics of
drug-induced protein interactions.
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Figure 6. Influence of NanoBiT on rapamycin-inducible FRB/FKBP interaction in cells. (a) Rapamycin potency (EC50) for inducing the interaction
between FRB-11S and FKBP fused to various peptides. (b) Induction of protein interactions with 30 nM rapamycin in HEK293T cells coexpressing
FRB-11S and FKBP-peptide. For both panels: n = 4, variability displayed as SD.
Figure 7. Measuring inhibitor-induced BRAF/CRAF dimerization. (a)
BRAF-11S and CRAF-114 dimerization in HCT116 cells following 2 h
incubation with different BRAF inhibitors. Calculated EC50 values
shown in legend. (b) Washout experiment showing reversibility of
BRAF-11S/CRAF-114 interaction. HEK293T cells were treated with 1
μM of the reversible inhibitor, GDC 0879, or the covalent inhibitor, AZ
628, for 2 h. For washout samples, inhibitor was removed by media
exchange and luminescence was measured 1 h later. Data represent the
remaining luminescent signal normalized to t = 0 of the curve fit. For
both panels: n = 4, variability displayed as SD.
Summary. NanoBiT is a complementation reporter designed
for quantitative investigation of protein interaction dynamics
under relevant physiological conditions. Benefiting from the
small size and bright luminescence of NanoLuc, it provides
detection at low intracellular concentrations with minimal steric
interference on appended target proteins. Notably, one of the
NanoBiT componenents is only 11 amino acids long,
comparable to other peptide tags routinely used in protein
analysis. The other NanoBIT component is also relatively small
in terms of protein tags (e.g., two-thirds the size GFP).
NanoBiT is distinct from other complementation reporters
because its components are structurally optimized for high
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conformational stability and low intrinsic affinity. Increased
stability designed into the large component (11S) provides
improved expression and reporter performance at physiological
temperature. Low intrinsic affinity, achieved by adapting the
small peptide component (114), minimizes its influence on the
interaction characteristics of the target proteins. The intrinsic
affinity and association constants determined for NanoBiT were
found to be outside of the ranges typical for protein interactions.
Thus, the luminescent signal from NanoBiT should provide an
accurate indication of interaction dynamics (e.g., for proteins
having KD < 10 μM), although caution is prudent when working
with unusually weak or transient interactions.
This capability was verified using SME1 in combination with a
set of interacting inhibitory proteins (BLIP) of differing affinities.
Appending the reporter had negligible influence on binding
affinity, although as with any fusion tag, the possibility of steric
interference for certain protein pairs should be considered.
Nonetheless, the luminescent signal from NanoBiT correlated
precisely with the independent assessment of protein association
through SME1 activity. In mammalian cells, the luminescence
produced by NanoBiT is well behaved and reflective of the
interaction status of the reporter. For the interaction of FKB/
FRBP, it showed second-order behavior consistent with a
dynamic binary system, while at higher concentrations favoring
full association of the dimeric complex, it conformed to the linear
characteristics of NanoLuc. The luminescent response was rapid
and reversible without evident lag, indicating that NanoBiT can
react nearly instantaneously to changing intracellular conditions.
Furthermore, dynamic processes can be monitored for more
than an hour due to the stable signal duration of the luminescent
reaction.
Our analysis indicates that NanoBiT fulfills the general
expectation of a complementation reporter in mammalian cells,
with biomolecular parameters generally suited for accurate
portrayal of intracellular processes. In addition to analyzing
protein interactions within cells, we also confirmed the ability to
characterize pharmacological outcomes. Specifically, NanoBiT
can reveal drug potency for induced protein interactions and the
binding kinetics underlying drug action. Moreover, with the
ability to substantially alter both size and intrinsic affinity of the
peptide component in this binary reporter, we believe this
luminescent system will provide additional applications yet to be
realized.
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■ METHODS
DNA Constructs, Protein Purification, and Lysate Prepara-
tion. See the Supporting Information.
Nluc Dissection and Screening. CP Nluc variants were made
using splicing by overlap extension (SOE).22 Each variant was designed
so that former termini were joined by a 33-amino acid polypeptide linker
containing a TEV protease sequence (underlined):
GSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGG. Variants were
screened by incubating bacterial, wheat germ, or mammalian lysates
with or without ProTEV Plus in 1X ProTEV buffer (Promega) at 30 °C
for 60 min. Samples were diluted in DMEM and then further diluted 1:2
in Nano-Glo Luciferase Assay Reagent (Promega) for 5 min at ambient
temperature before measuring luminescence with a GloMax-Multi+
Detection System (Promega).
Sequence Optimization. DNA encoding Nluc amino acids 1−156
was mutated by error-prone PCR (Diversify PCR Random Mutagenesis,
Clontech), subcloned into pF4Ag,9 and used to transform KRX E. coli.
Lysates of 3,696 variant clones were screened (Freedom robotic
workstation (Tecan)) by incubation with HaloTag-NP (KRX lysates)
for 10 min with shaking. Nano-Glo Luciferase Assay Reagent was added,
and luminescence was measured on a GENiosPro reader (Tecan). In
addition to fully random mutagenesis, we attempted to engineer
structural stability to 156 by reducing conformational entropy.23 Each
glycine residue was substituted to alanine, and beneficial (∼1.5-fold
improved luminescence) changes (G15A, G35A, G51A, G67A, and
G71A) were combined with mutations identified from the random
library screen. The combined variant providing the highest
luminescence was used as the template for a second library, where
11,088 variants were screened in similar fashion. Improved variants
identified during primary screening were validated using a secondary
screen, where clones were processed the same way but in triplicate.
Verified hits were then sequenced.
Synthetic peptide arrays (≥75% purity; N-terminus acetylated and
the C-terminus amidated; New England Peptide) were designed to
semirandomly sample combinatorial sequence space (via generally
conservative amino acid substitutions and deletions at each terminus)
across NP. Peptides were dissolved in water and concentration
quantified by A280. All other synthetic peptides of interest were
synthesized at >95% purity (Peptide 2.0). Relative binding affinity and
maximal luminescence were determined by titrating the peptide into
large NanoBiT variants as described below under peptide affinities.
Intracellular and Thermal Stability. HeLa cells expressing 156,
11S, or Nluc were plated at 104 cells/well (96-well plate, Corning), and
24 h later medium was exchanged with growth medium containing 0.4
mM cycloheximide or DMSO. Cells were assayed over 6 h by addition of
Nano-Glo Luciferase Assay Reagent (with 100 μM NP for 156 and 11S)
and measured on a GloMax-Multi+ Detection System. Cycloheximide-
treated samples were normalized to DMSO-treated samples for each
time point, and data were fit (one phase decay) using GraphPad Prism 6.
Thermal stability analysis was carried out by incubating 1.5 pmol 156
or 30 fmol 11S (in DMEM containing 0.1% Prionex) for 30 min at
different temperatures. Following rapid cooling to 4 °C, 300 pmol of
synthetic NP was added, and samples were incubated at 22 °C for 5 min.
Nano-Glo Luciferase Assay Reagent was added, and luminescence was
measured using an Infinite F500 reader (Tecan). Activity was
normalized to control samples incubated at 4 °C. Tm represents the
temperature at which 50% of protein is inactive.
Specific Activity. Purified Nluc, 156, and 11S were diluted to 40 pM
in assay buffer (PBS pH 7, 0.01% Prionex, 0.005% Tergitol, 1 mM
DTT). Synthetic NP was titrated (64 nM−140 μM for 156; 9 nM−20
μM for 11S; buffer in place of peptide for Nluc) and samples assayed
with variable furimazine (Fz) concentrations (78 nM−10 μM for Nluc;
625 nM−20 μM for 156/11S) in Nano-Glo Buffer. For each
concentration, maximal luminescence (RLUmax) was calculated using
the equation,
RLUmax × [Peptide]
RLU =
KD + [Peptide].
Vmax was then calculated using the equation,
407
Articles
RLUmax =
Vmax × [Fz]
KM + [Fz].
Peptide Affinities. Purified 11S (40 pM) or 156 (400 pM) were
mixed with various concentrations of synthetic peptide (Peptide 2.0) in
assay buffer and incubated at ambient temperature for 30 min. Nano-
Glo Luciferase Assay Reagent was added and luminescence measured on
a GloMax-Multi+ Detection System. Average RLUs over 10 min were fit
(one site specific binding) using GraphPad Prism 6, and the fits were
subsequently used to determine KD values.
β-Lactamase Assays. Purified BLIP variants were incubated with
SME1 for 2 h at ambient temperature. Nitrocefin (EMD Millipore) was
added to the equilibrated complex, and substrate hydrolysis was
monitored (A280) every 25 s for 25 min on an Infinite M1000 reader
(Tecan). Using these measurements, inhibition constants were
calculated as previously described.13
Luminescence-based Binding Assays. KD values were deter-
mined by adding Fz (10 μM) to the equilibrated complex, measuring
luminescence on a GloMax-Multi+ Detection System every 1 min for 30
min, and fitting the data to the equation,
RLUmax × [BLIP]
RLU =
KD + [BLIP].
For BLIP binding rate constants, BLIP-114, SME1-11S, and Fz were
combined, and luminescence was monitored for 15 min (BLIP WT-
114) using a GloMax-Multi+ Detection Systemor every 1 s for 100 s
(BLIP Y50A-114 and BLIP R160A-114) using the Infinite M1000
reader (Tecan). Observed rate constants (kobs) were determined by
fitting (one phase association) with GraphPad Prism 6. The observed
rate constant was then plotted versus BLIP-114 concentration, and the
linear fit was determined. The association and dissociation rate
constants were determined by fitting kobs data to the equation,
kobs = kon × [BLIP] + koff .
Intracellular Luminescence. For each model, the eight possible
orientations of the reporter subunits fused onto the interacting proteins
were screened for maximal S/B. Using the optimal orientations,
mammalian cells transiently expressing the fusion proteins (1:1 ratio of
interacting pairs) were plated at 2 × 104 cells/well (96-well plate,
Corning) and incubated ± drug at 37 °C. For FRB/FKBP, HEK293T
cells were incubated for 2 h with rapamycin (EMD Millipore) in Opti-
MEMI. For BRAF/CRAF, HEK293T or HCT116 cells were serum
starved for 4 h in Opti-MEMI before incubation for 1 h with GDC 0879
(R&D Systems), AZ 628 (R&D Systems), Sorafenib (Cell Signaling
Technology), or PLX4720 (Cayman Chemical Company). Nano-Glo
Live Cell Substrate (Promega) was added to a 1× concentration, and
luminescence was measured using a GloMax-Multi+ Detection System.
For kinetic measurements, HEK293T cells were plated at 1 × 104
cells/well and transfected the following day with ADRB2, AVPR2,
ARRB2, PRKAR2A, or PRKACA DNA. Twenty-four hours post-
transfection, growth medium was exchanged with Opti-MEMI, and cells
were incubated for 3.5 h at 37 °C. Nano-Glo Live Cell Substrate was
added, and luminescence was measured using a Varioskan Flash
Multimode Reader (Thermo Fisher Scientific) at either 21, 28, or 37 °C.
Following thermal equilibration, compound was added by injection to
final concentrations of 10 μM (isoproterenol, propranolol, forskolin) or
100 nM (Arg8-vasopressin) in Opti-MEMI and measurements
continued. Split Fluc assays were performed in the same manner except
using 2 mM Luciferin-EF (Promega). The GloSensor cAMP Assay
(Promega) was used to monitor the cAMP response following the
manufacturer’s recommended protocol at variable temperatures and
with the addition of 2% (v/v) GloSensor cAMP Reagent 30 min prior to
beginning luminescence measurements.
Curve fitting luminescence linearity in cells and lysates was modeled
using the expected first order relationship between Nluc expression and
RLUs (a[DNA]+b) and the expected second order relationship for
NanoBiT (a[DNA]2/(k+[DNA])+b).
Additional methods can be found in the Supporting Information.
DOI: 10.1021/acschembio.5b00753
ACS Chem. Biol. 2016, 11, 400−408
ACS Chemical Biology Articles

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*
ASSOCIATED CONTENT
S Supporting Information
(11) Paulmurugan, R., and Gambhir, S. S. (2007) Combinatorial
library screening for developing an improved split-firefly luciferase
fragment-assisted complementation system for studying protein-protein
The Supporting Information is available free of charge on the interactions. Anal. Chem. 79, 2346−2353.
ACS Publications website at DOI: 10.1021/acschem- (12) Swanson, B., Fan, F., and Wood, K. V. (2007) Enhanced response
bio.5b00753. dynamics for transcription analysis using new pGL4 luciferase reporter
Figures s1−s18, Tables s1−s5, and supporting methods vectors. Cell Notes 17, 3−5.
(13) Zhang, Z., and Palzkill, T. (2003) Determinants of binding affinity
(PDF) and specificity for the interaction of TEM-1 and SME-1 beta-lactamase

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with beta-lactamase inhibitory protein. J. Biol. Chem. 278, 45706−45712.
AUTHOR INFORMATION (14) Petrosino, J., Rudgers, G., Gilbert, H., and Palzkill, T. (1999)
Contributions of aspartate 49 and phenylalanine 142 residues of a tight
Corresponding Author binding inhibitory protein of beta-lactamases. J. Biol. Chem. 274, 2394−
*Tel.: 608-274-1181. E-mail: lance.encell@promega.com. 2400.
Present Address (15) Oakley, R. H., Laporte, S. A., Holt, J. A., Barak, L. S., and Caron,
§ M. G. (1999) Association of beta-arrestin with G protein-coupled
Department of Pharmaceutics and Pharmaceutical Chemistry,
receptors during clathrin-mediated endocytosis dictates the profile of
University of Utah, Salt Lake City, Utah 84112, United States
receptor resensitization. J. Biol. Chem. 274, 32248−32257.
Notes (16) Binkowski, B. F., Butler, B. L., Stecha, P. F., Eggers, C. T., Otto, P.,
The authors declare no competing financial interest. Zimmerman, K., Vidugiris, G., Wood, M. G., Encell, L. P., Fan, F., and

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Wood, K. V. (2011) A luminescent biosensor with increased dynamic
ACKNOWLEDGMENTS range for intracellular cAMP. ACS Chem. Biol. 6, 1193−1197.
(17) Ohmuro-Matsuyama, Y., Chung, C. I., and Ueda, H. (2013)
We thank G. Vidugiris for help with screening sequence libraries Demonstration of protein-fragment complementation assay using
and G. Colwell at Gene Dynamics, LLC, for assistance with purified firefly luciferase fragments. BMC Biotechnol. 13, 31.
vector constructions. (18) Banaszynski, L. A., Liu, C. W., and Wandless, T. J. (2005)

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Characterization of the FKBP-rapamycin-FRB ternary complex. J. Am.
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408 DOI: 10.1021/acschembio.5b00753

ACS Chem. Biol. 2016, 11, 400−408