Two-state selection of conformation-
Junjun Gaoa, Sachdev S. Sidhu1,b, and James A. Wellsa,2
aDepartments of Pharmaceutical Chemistry and Cellular and Molecular Pharmacology, University of California, 1700 4th Street, San Francisco, CA 94143;
andbDepartment of Protein Engineering, Genentech, Inc, 1 DNA Way, South San Francisco, CA 94080
Communicated by Robert M. Stroud, University of California, San Francisco, CA, December 22, 2008 (received for review October 19, 2008)
We present a general strategy for identification of conformation-
specific antibodies using phage display. Different covalent probes
were used to trap caspase-1 into 2 alternative conformations,
forms of the protease were used as antigens in alternating rounds
of selection and antiselection for antibody antigen-binding frag-
ments (Fabs) displayed on phage. After affinity maturation, 2 Fabs
were isolated with KD values ranging from 2 to 5 nM, and each
their noncognate conformer. Kinetic analysis of the Fabs indicated
that binding was conformation dependent, and that the wild-type
caspase-1 sits much closer to the off-form than the on-form.
Bivalent IgG forms of the Fabs were used to localize the different
states in cells and revealed the activated caspase-1 is concentrated
in a central structure in the cytosol, similar to what has been
described as the pyroptosome. These studies demonstrate a gen-
eral strategy for producing conformation-selective antibodies and
show their utility for probing the distribution of caspase-1 confor-
mational states in vitro and in cells.
allostery ? caspase-1 ? phage display ? protein conformational change
selection upon binding of different small molecules, biopoly-
mers, or metal ions or through posttranslational modifications.
Structural methods give us high-resolution insight into the
nature of these conformational transitions in vitro but have
limited use for determining the equilibrium distribution of these
states in solution or in cells. To expand the tools useful for
trapping and analyzing conformational states in enzymes, both
in solution and in cells, we developed a general strategy for
As a test case, we focused on caspase-1, an aspartate-specific
thiol protease that is critical for processing of proinflammatory
cytokines during the innate immune response (for review, see
exists primarily as a monomer in solution (4, 5). Upon innate
immune stimuli, the proenzyme is believed to dimerize by
binding to scaffolding proteins known collectively as the inflam-
masome. This triggers proteolytic autoactivation or transactiva-
tion, in which the propeptide and an intersubunit linker are
cleaved (6, 7). Crystal structures of the mature protease with
various small molecules bound show that it can exist in at least
2 conformations (8–10). When an active site inhibitor is bound,
the enzyme appears to be in a catalytically competent form
(called the on-form) (9). However, binding of covalent disulfide
ligand to a central cavity ?15 Å from the active site stabilizes the
protease in an inactive state (called the off-form) (8). This
allosterically inhibited state is virtually identical to the apo-form
of the enzyme as seen in the crystal structures. The dimeric
enzyme shows positive cooperativity [nhill? 1.5 (8)], and mu-
tational studies reveal that only a small set of residues (a
‘‘hot-wire’’) mediates the on-to-off transition between the allo-
rotein allostery is a central means to regulate protein func-
tion in cells. Allostery is mediated through conformational
steric and active sites (11). These data support a dynamic
activation model for caspase-1 (Fig. 1A).
We wished to generate monoclonal antibodies to each of the
on- and off-states to better understand the equilibrium distri-
bution of these states in solution and in cells and to provide
probes to localize these forms in cells. We trapped homogeneous
forms of the on- and off-states of caspase-1 using the active site
inhibitor (Ac-YVAD-cmk) to lock the on-state and compound
34 (1-methyl-3-trifluoromethyl-1H-thieno[2,3-c]pyrazole) to
lock the off-state. These conformation-locked forms of
caspase-1 were then used as antigens in sorting codon-restricted
phage display libraries (12) to generate high-affinity antibody
Author contributions: J.G., S.S.S., and J.A.W. designed research; J.G. performed research;
S.S.S. contributed new reagents/analytic tools; J.G., S.S.S., and J.A.W. analyzed data; and
J.G., S.S.S., and J.A.W. wrote the paper.
The authors declare no conflict of interest.
1Present address: Banting and Best Department of Medical Research and the Donnelly
Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON,
Canada M5S 3E1.
2To whom correspondence should be addressed: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2009 by The National Academy of Sciences of the USA
activation of caspase-1. It has been suggested that in cells procaspase-1 exists
primarily as monomer. Upon binding to scaffolding proteins (NALPs, ASC,
etc.), procaspase-1 dimerizes and undergoes proteolytic activation. Mature
caspase-1 is in equilibrium between off- and on-conformations. Binding of
ligands at the active or allosteric site can shift the equilibrium toward the on-
or off-state. (B) Covalent labeling of apo-caspase-1. Irreversible active-site
inhibitors or allosteric compounds were used to trap caspase-1 into a stable
conformation for antibody selection.
Model and labeling design. (A) Proposed model for the dynamic
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fragments (Fabs) that were selective for either the on- or the
off-state. The combination of small molecules to trap different
molecular conformations coupled with in vitro selection pro-
vides a general approach to produce multiple conformation-
selective antibodies to biomolecules.
Selection of Conformation-Specific Fabs Using Phage Display. A
major challenge to produce conformation-selective antibodies
against caspase-1 is to have relatively homogenous preparations
of each conformer. To lock caspase-1 in the off-state, we labeled
the enzyme with a thiol-containing compound 34 that covalently
bound to Cys-331 on the small subunit within the allosteric
pocket (8). Similarly, an irreversible active-site inhibitor Ac-
YVAD-cmk (CalBiochem) was used to trap the on-state (Fig.
1B). The stoichiometric labeling by the different covalent inhib-
itors to thiols in either the active site or allosteric site was
confirmed by LC-MS (Fig. S1).
We used a phage-display strategy that allowed us to maintain
the state of antigen throughout the selection process in vitro
(Fig. 2). Two synthetic libraries were built on a single antibody
fragment framework by introducing limited diversity into the 3
heavy chain CDRs (Complementarity-determining regions:
CDR-H1, -H2, and -H3) and the third CDR of the light chain
(CDR-L3) (13, 14). A Tyr/Ser focused library was used that
explicitly placed Tyr and Ser at solvent accessible positions
responsible for antigen recognition (14). A focused Tyr/Ser/Gly/
Arg library added further chemical diversity by incorporating
Gly and Arg to the binary Tyr/Ser background in CDR-H3 (25%
Tyr, 25% Ser, 25% Gly, 25% Arg) (13). The combined naïve
library pools contained ?1010unique clones.
antigens. After the first 2 rounds, antiselection was enforced by
preincubating the amplified phage pool with the off-target
conformer. Ninety-six colonies were tested by spot phage ELISA
after round 6, and clones showing ?20-fold selectivity (based on
OD450for the target conformer/OD450for the off-target con-
former) were discarded. To exclude Fabs that bound to the
active-site or allosteric inhibitors, a second ELISA screen was
performed by using a different set of inhibitor-trapped caspase-1
(z-WEHD-fmk for trapping the on-form and compound 11 for
trapping the off-form). Only Fabs that showed high selectivity
and that bound independently of the specific inhibitors were
sequenced. Affinities of Fabs on selected phage were measured
by solution-competition phage ELISA (15, 16) (Table S1 ).
Two different affinity maturation strategies were undertaken
to further improve the Fabs (Fig. 2). We focused first on
optimizing Fabs for the on-form by partial randomization of all
3 CDR loops on the heavy chain (17). Four clones with affinities
ranging from 50 to 110 nM were used as independent starting
templates (Table S1). We found that a single amino acid change
(M to T) at position 100c in the CDR-H3 resulted in the biggest
improvement in affinity (?20-fold). The tightest binder (called
Fabon) was chosen for expression and subsequent analysis (Table
1). The same strategy failed to improve the affinity of Fabs for
the off-form. Therefore, we shifted our attention to the light-
chain CDR loops by randomizing these sequences based on the
Kinetic analysis was performed by immobilizing different forms of caspase-1
was used to fit the recorded sensograms and determine the kinetic parame-
ters. (B) The relative difference in KDof Fabs binding to different forms of
caspase-1 was calculated from data in A.
Fabs are conformation-selective. (A) SPR analysis of Fabs by BIAcore.
spot phage ELISA. Positive clones that showed both good affinity and selec-
out for affinity maturation. Fabs and IgGs were expressed and purified in E.
Selection for conformation-selective antibodies. Six rounds of phage
Table 1. Sequences of Fab clones after affinity maturation
29 30 31
3350 51 52a
Only CDR sequences at positions that were randomized in the libraries are shown. The numbering is according to the nomenclature of Kabat et al. (34).
www.pnas.org?cgi?doi?10.1073?pnas.0812952106 Gao et al.
natural diversity of human kappa light chain sequences in the
Kabat database (18, 19). This resulted in ?100-fold improve-
ment of affinity for the best clone (called Faboff, Table 1).
Biochemical Characterization of Conformation-Specific Antibodies.
We expressed Fabonand Faboffin Escherichia coli and purified
them by protein-A affinity chromatography. To characterize the
binding affinity and selectivity of the Fabs, we tested their
interaction against various caspase-1 conformers by surface
plasmon resonance (SPR). As shown in Fig. 3A, both Fabs bind
to their target conformers tightly with low-nM KDvalues that
were similar to those estimated from solution-competition phage
ELISA (12.9 ? 1.1 nM for Fabon-phage; 7.9 ? 0.5 nM for
Faboff-phage). Fabon showed no binding to the off-state
caspase-1 even at highest test concentration (1 ?M) on BIAcore,
whereas Faboffbound to the on-state caspase-1 at least 20-fold
weaker than to its cognate form (99 vs. 4.7 nM, respectively). The
by the difference in konnot koff. This suggests these Fabs are
selective for the specific conformation of caspase-1.
Given the sensitivity of the Fabs for different caspase-1
conformers, we were intrigued to investigate the conformational
state of ligand-free caspase-1. Kinetic analysis using BIAcore
showed that Faboffbound to apo caspase-1 with nearly 20-fold
higher affinity than Fabon(17 vs. 330 nM, respectively; Fig. 3A).
Again, most of the difference was in kon, not in koff. Comparing
how well ligand-free caspase-1 bound to both Fabs with that of
the locked-on and -off forms (Fig. 3B), we concluded that
caspase-1 is in an equilibrium between the on- and off-states but
that the dominant species is much closer to the off-state con-
Activation or Inhibition of Caspase-1 with Conformation-Specific
Antibodies. We explored the possibility of affecting caspase-1
activity by stabilizing each of the 2 conformational states upon
binding to the 2 different Fabs. As seen in Fig. 4A, Fabon
increased the catalytic activity of caspase-1 by over 3-fold in a
dose-dependent manner. The EC50value (11.7 ? 2.2 nM) was
close to its KDfor the fully locked on-state conformer (2.5 nM,
Fig. 3A). In contrast, Fabofffunctioned as an inhibitor, which
prevented substrate binding by trapping the free enzyme in the
off-state. The EC50and IC50for activation and inhibition were
28-fold lower or 54-fold higher than their respective KDs for
binding of the apo-caspase-1 to Fabonor Faboff(Fig. 4B). This is
consistent with previous studies showing that the enzyme ex-
hibits positive cooperativity (8, 11), where substrate drives the
enzyme from the off- to the on-form (Fig. 1A).
Effects on Binding to Procaspase-1 in Vitro and in Cells. Previous
biochemical studies have suggested that procaspase-1 exists as a
monomer (4, 5). To determine the binding of the conformation-
specific antibodies to procaspase-1, we expressed and purified a
recombinant form of procaspase-1 in which the CARD domain
was deleted, called p32 (residues 120–404). SPR analysis showed
converted Fabs into full-length human dimeric IgGs and mea-
sured their binding to p32 and to ligand-free caspase-1. Both
IgGs bound to ligand-free caspase-1 like their Fab counterparts;
however, the binding affinity of IgGoff was at least 500-fold
stronger than IgGon(Fig. 5A). The dimeric IgGoffwas able to
bind to p32 with an affinity of 130 nM, possibly by driving the
dimerization of p32. Even though we cannot exclude the possi-
bility that the binding epitope of IgGs might include the cleavage
site, which is present in the mature form but not in the pro form,
it is less likely because both antigens (on- and off-form
caspase-1) we used in the selection had the same cleavage site
present. That IgGoncould not bind but IgGoffsuggests p32 exists
in a state much closer to the off-state.
To test whether IgGoffrecognizes full-length procaspase-1 in
cells, we carried out immunoprecipitation experiments in cell
extracts from human THP-1 cells that have high levels of
endogenous procaspase-1. As depicted in Fig. 5B, both IgGon
and IgGoffpulled down caspase-1, whereas only the latter was
able to immunoprecipitate full-length procaspase-1. These re-
sults align with the binding data above and further suggest that
the IgGs are useful tools for studying the specific conformations
of caspase-1 in cells.
nM) was preincubated with serial dilution of Fabs before assaying with
fluorescent substrate Ac-WEHD-fmk at 100 ?M concentration. The relative
change in enzyme velocity was calculated by dividing the enzyme velocity at
any given Fab concentration with the average velocity of enzyme in the
absence of Fab. Nonlinear regression was used to fit the curve and calculate
EC50and Ki. (B) The ratios of KD(apo caspase-1)for each Fab to the corresponding
EC50or IC50were calculated from data in A and Fig. 3A.
Activation or inhibition of caspase-1 activity by Fabs. (A) Caspase-1 (5
on the CM5 chip and injecting serial dilutions of IgGs. BIAevaluation software
was used to fit the recorded sensograms and determine the kinetic parame-
ters. (B) Immunopreciptation of procaspase-1 and apo caspase-1 from THP-1
lysates by IgGs. Monoclonal anti-caspase-1-p10 antibody (Calbiochem) that
recognizes both procaspase-1 and caspase-1 p10 subunit was used in Western
IgGoffbinds to procaspase-1. (A) SPR analysis of IgGs by BIAcore.
Gao et al.
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To investigate whether the antibodies have similar effects on
caspase-1-mediated cleavage in cells, we examined the process-
ing of pro-IL-1ß, a well characterized natural caspase-1 sub-
strate, in the presence of each of the 2 IgGs. THP-1 cells were
stimulated by LPS for 4 h to increase pro-Il-1ß expression, and
cell extract was prepared as described (7). Western blots probed
for mature Il-1ß showed that IgGoff significantly blocked
caspase-1-mediated cleavage of pro-Il-1ß (Fig. 6). IgGon also
inhibited processing of pro-IL-1 ß but to a lesser extent possibly
by sterically blocking the larger protein substrate binding. These
results suggest that IgGoffcan effectively inhibit caspase-1 ac-
tivity in cells by sequestering the enzyme in the off-state.
Localization of Active Caspase-1 Conformer in Cells. The ability of
IgGonto bind selectively to the on-form of caspase-1 and not to
procaspase-1 makes it potentially useful for tracking caspase-1
activation in cells. THP-1 cells can be differentiated into mac-
rophages with PMA treatment, which then become adherent to
glass coverslips. After fixation and mild detergent permealiza-
tion, IgGonwas added as the probe for the on-state of caspase-1.
Immunofluorescent microscopy revealed that most of cells stim-
ulated with LPS/ATP showed a general increase in diffuse
fluorescent signal (Fig. S3). Intriguingly, ?10% of these cells
contained a single bright, near-spherical structure located in the
cytosol (Fig. 7). That we observed this structure only when
staining with IgGon and not with IgGoff suggests it is highly
enriched for the on-state of caspase-1.
We present a general strategy for directly obtaining conforma-
tion-selective antibodies that was applied to study distributions
of on- and off-conformers of caspase-1. The differences in
conformation for the on- and off-states as viewed by X-ray
crystallography are relatively modest. Thus, that high-affinity
antibodies specific for the 2 states could be isolated suggests the
wide applicability of this strategy.
The key elements of this approach are the availability of
ligands to trap specific conformations of the protein of interest
coupled with an in vitro selection method to isolate specific
binding proteins. Although we used covalent ligands to lock
specific conformations, one could also employ noncovalent
ligands, so long as one worked at a high enough concentration
to maintain saturation. It should also be possible to trap these
alternate target conformations with natural protein-binding
partners or using engineered disulfide or chemical cross-linkers.
State-selective binding partners have been identified before. It
has been reported that using phage display to isolate peptides
specific for estradiol- or tamoxifen-activated estrogen receptor
(20). Fabs have also been selected for GTP-bound Ras (21).
However, neither study selected for more than 1 state. Recent
elegant work by Grutter and coworkers (22) has shown that it is
possible to isolate conformation-specific binding proteins to
caspase-2 without using conformation-trapped antigens. How-
ever, this approach required the assaying of ?100 individual
clones following selections from a combinatorial library of
designed ankyrin repeat proteins (DARPin) to identify the
In vitro selection allowed antiselection in solution (23) with
the noncognate form of the protein, which enabled the dramatic
enrichment of selective binders. Although we found phage
display to be particularly effective in vitro selection, there are a
variety of other methods one could apply, including ribosome
display, yeast display, and others (for review, see refs. 24 and 25).
Fab display is particularly effective, because it is well established
for naïve selections and has been made even more effective
recently by using codon-restricted CDR libraries enriched for
Ser and Tyr residues (12, 14). However, there is a variety of other
scaffolds that could be potentially used, such as DARPins (26),
Affibodies (27), fibronectin type III domains (28), zinc finger
DNA-binding domains (29), and many others. Moreover, one
could imagine using this approach to identify conformation-
selective antibodies to other biopolymers such as alternate forms
of DNA or RNAs such as riboswitches, etc.
Our data suggest that the Fabs we identified are specific for
the conformers of caspase-1 they were selected for. First, at the
selection stage, we found strong enrichments for the specific
antigen used in the selection, and binders survived the antis-
election steps with noncognate antigens. The consensus se-
THP-1 cell extracts were incubated with IgGs at 4 °C overnight and probed
by Western blot analysis using an antibody specific for mature IL-1ß (Cell
Inhibition of caspase-1 by IgGoffin THP-1 extracts. LPS-stimulated
in C. Note the spherical body found in ?10% of cells.
Probing active conformers of caspase-1 in THP-1 cells. LPS-stimulated THP-1 cells were stained with DAPI (A) and IgGon(B). The merged image is shown
www.pnas.org?cgi?doi?10.1073?pnas.0812952106Gao et al.
quences for the Fabonand Faboffwere very different from each
other, and they bound to their cognate antigen with 20- to
500-fold higher affinity than to the noncognate forms. Strong
evidence for conformational specificity was provided by the SPR
data, which showed that the increased affinity for the cognate
antigen was due to a faster on-rate rather than a slower off-rate.
Interestingly, the fact that the antibodies can bind to the
noncognate form at all suggests each of the inhibitor-locked
antigens is dynamic, albeit the inhibitor-locked cognate con-
former is much more populated than the noncognate conformer.
In addition, both Fabs did not cross-react with caspase-4 and -5,
which have the highest sequence similarity to caspase-1 (Fig. S4).
We do not know the exact epitopes where these Fabs bind on
caspase-1. However, it is very unlikely their epitopes include the
inhibitor itself. In fact, the structure of the inhibitor was alter-
nated during positive selection to avoid antibodies that inter-
acted directly with the inhibitor.
suggests strongly that the enzyme in solution is dynamically
interchanging between conformations because the affinity for
Fabs is intermediate between the locked forms of caspase-1 (Fig.
3B). However, the apo enzyme sits overwhelmingly in the
off-form. For example, the enzyme binds with an affinity that is
much closer to the locked off-form of caspase-1 than to the
on-form. The ratios of KDvalues for the Faboffto Fabonvary ?4
logs; the unlabeled caspase-1 is within 1 log unit of the locked-off
form yet 3 log units away from the locked-on form of caspase-1.
Thus, the average ensemble of conformations for apo caspase-1
is intermediate between the on- and off-states but populates the
off- over the on-state by a factor of ?1,000. It is noteworthy that
X-ray structures show the conformation of the apo-form of
caspase-1 (9) is virtually identical to the off-form generated by
the allosteric tethered ligand (8).
The Fab fragments have marked functional effects on the apo
caspase-1 that are consistent with previous reports that the
enzyme exhibits positive cooperativity (8, 11). The titration of
apo-caspase-1 with Faboncauses ?3-fold activation of activity
(Fig. 4A) probably by binding and stabilizing the active con-
former. Moreover, the EC50for activation by Fabon(11 nM) is
significantly lower than the KDfor binding (300 nM; Fig. 4B).
These data suggest that the presence of substrate can stabilize
the active conformer and thus improve apparent affinity. Like-
is significantly above the KDfor binding of Faboff(5 nM) and
reflects the fact that substrate stabilizes the on-form and thus
competes for binding of Faboff. We previously showed that
labeling caspase-1 with Ac-YVAD-cmk and the allosteric inhib-
itor are mutually exclusive: binding of the active site inhibitor
promotes a conformation that is incompatible with binding of
the allosteric inhibitor and vice versa (8). Similarly the Fabs
appear to bind to mutually exclusive conformations and away
from these inhibitor binding sites. Thus, the enzyme can be
allosterically activated or inhibited through binding of confor-
mation-specific antibodies on different surfaces.
One of the advantages of using Fabs is that they are readily
converted into IgGs that allow one to study possible avidity
effects. Interestingly, the IgGoff bound 40 times tighter than
Faboffto apo-caspase-1, whereas IgGonbound with nearly iden-
tical affinity to Fabon. This may reflect the different position of
the binding epitope on caspase-1 and resultant stoichiometry
such that 1 IgGoff molecule can bind to both subunits of
caspase-1 simultaneously, whereas 1 IgGonmolecule cannot. The
enhancement in affinity seen for IgGoff for Faboff is almost
entirely due to enhanced on-rate, which may reflect that
the dimer has a greater probability of productive binding per
Previous studies indicate that procaspase-1 is monomeric (4,
30), yet very little is known about the conformation of this form
in vitro or in cells. We find the IgGoff binds modestly to
procaspase-1 but neither IgGon nor Fabon nor Faboff bind
detectably (Fig. 5A). This suggests that the pro-form is much
closer to the off-form of caspase-1 in conformation and that
dimerizing procaspase-1 by binding to IgGoffpromotes a con-
formation more like that of apo caspase-1. Nonetheless, the
pro-form has a conformation that is not identical to the off-form
because IgGoff bound to procaspase with a KD of 130 nM
compared with binding to caspase-1 with a KDof 0.38 nM. These
results were mirrored in the immunoprecipitation experiments
(Fig. 5B). The IgGoff can pull down both procaspase-1 and
caspase-1 in THP-1 cells, whereas IgGoncan pull down only the
The additional advantage for converting the Fabs to IgGs was
that these can be used with standard immunostaining reagents
to localize specific conformations of caspase-1 in cells. In THP-1
cells probed with IgGoff,we saw only diffuse staining of the cells
whether stimulated with LPS or not (data not shown). In
contrast, when THP-1 cells were treated with LPS and stained
with IgGon, we found intense staining of a single supermolecular
cytosolic structure (1?2 ?m in diameter) in ?10% of the cells.
Alnemri and coworkers have reported the existence of a virtually
identical supermolecular structure, which they termed the py-
roptosome, in 15–30% of THP-1 cells stimulated with LPS using
GFP-tagged ASC (apoptosis-associated speck-like protein con-
taining a CARD domain) (30). That we can stain the supermo-
lecular structure only with IgGonnot IgGoffsuggests strongly that
virtually all of the caspase-1 is processed to the mature form and
rests in the active conformation in the pyroptosome-like struc-
ture. This would indicate that caspase-1 is either actively cata-
scaffold stabilizes the on-form of caspase-1. In this regard, ASC
may function like the on-state antibody; however, its binding site
does not overlap with that of IgGon.
Overall, these studies demonstrate a general approach for
selecting binding proteins to specific protein conformations
using small molecule ligands to lock conformations of interest
and phage display to identify conformation-selective antibodies.
The Fabs and IgG derivatives were useful for defining and
localizing the specific forms of caspase-1 in vitro and in cells.
Moreover, this approach could be useful for generating specific
inhibitors or even activators of proteins in cells extracts and
possibly in cells by using appropriate intracellular antibody
delivery technology (31, 32).
Materials and Methods
Caspase-1 Expression and Purification. The p20 subunit (residues 120–279) and
expressed in E. coli as inclusion bodies from a pRSET expression vector (In-
performed as described (8). The Cys285Ala mutant of caspase-1 was made by
of procaspase-1 lacking the CARD domain (CARDless procaspase, residues
120–404) was cloned into a pET23b expression vector (Novagen) with a
C-terminal His6tag and transformed into E. coli BL21(DE3) strain. The expres-
sion was induced with 0.2 mM IPTG induction for 20 min at OD600?0.6. Cell
pellets were lysed by 5 passes through a microfluidizer in ice-cold lysis buffer
(100 mM Tris, pH 8.0, 100 mM NaCl). The lysate was cleared by centrifugation
at 48,500 ? g for 15 min at 4 °C. The supernatant was first loaded on a 5-mL
HisTrap HP column (GE Healthcare), and bound protein was eluted with a 0-
mM Tris, pH 8.0, 5% glycerol, and loaded on a 5-mL HiTrap Q HP column. The
p32 was eluted with a 0- to 0.5-M NaCl gradient and aliquots were frozen
immediately in an ethanol-dry ice bath.
Caspase-1 Labeling. To prepare the on-form caspase-1, wild-type caspase-1
was incubated with 4-fold excess of active-site inhibitor (Ac-YVAD-cmk or
z-WEHD-fmk) at 4°C overnight in the labeling buffer (50 mM Hepes, pH 8.0,
200 mM NaCl, 50 mM KCl, 200 ?M ß-ME). Protein precipitate was removed by
centrifugation, and the labeling was confirmed by the mass shift observed by
Gao et al.
March 3, 2009 ?
vol. 106 ?
no. 9 ?
LC-MS (Waters). To prepare the off-form of caspase-1, a catalytic-inactive
caspase-1 Cys285Ala was incubated with 150 ?M of the allosteric inhibitor
[compound 34 or compound 11 (8)] at 4 °C overnight in the same labeling
buffer containing 1 mM ß-ME. For random biotinylation, the off-form of
caspase-1 was incubated with 15-fold excess sulfo-NHS-LC-biotin (Pierce) for
45 min at ambient temperature, and the reaction was stopped by buffer
exchange using a NAP-25 column (GE Healthcare).
Library Construction and Sorting. We modified the Fab-template phagemid
chain CDR-L3 to reduce wild-type Fab background. For the construction of
naïve libraries, the resulting phagemid was used as the ‘‘stop template’’ in a
mutagenesis reaction with oligonucleotides designed to repair simulta-
neously the stop codons and introduce designed mutations at the desired
sites, as described (16).
In sorting for on-form specific Fabs, the phage pool was cycled through
directly immobilized on 96-well Maxisorp plate (Thermo Fisher). Bound
phage were eluted with 100 mM HCl and neutralized with 1 M Tris, pH 8.0.
Phage were amplified in E. coli XL1-blue (Stratagene) with the addition of
M13-KO7 helper phage (New England Biolabs). In sorting for the off-form
specific Fabs, a solution-phase binding strategy was adapted for better
control over the selection and anti-selection process. The phage pool was
incubated for 2 h at room temperature with biotinylated allosteric con-
former before being captured on neutravidin or streptavidin (Pierce)
coated Maxisorp plates. The bound phage were then eluted and propa-
gated as described above. After selection, individual clones were picked
and grown in a 96-well deep well plate with 2YT broth supplemented with
carbenicillin and M13-KO7. The culture supernatants were used in phage
ELISAs to identify binding clones (33).
was converted into the Fab expression vector by deleting the sequence
encoding for the cP3 minor phage coat protein and inserting a ? terminator
sequence (GCTCGGTTGCCGCCGGGCGTTTTTTAT) downstream of the stop
codon at the end of CH1domain. Fab protein was secreted from E. coli 34B8
strain transformed with individual plasmids in low-phosphate medium at
30 °C for 26 h, as described (18). To generate IgG proteins, the variable
domains were subcloned into vectors designed for transient IgG expression in
CHO cells (18). Fab proteins were purified with protein A affinity chromatog-
Kinetic binding analyses were performed by surface plasmon resonance
(SPR) using a BIAcore 3000 (GE Healthcare). Ligand-bound or free caspase-1
dimers were immobilized on CM5 chips and serial dilutions of Fabs or IgGs
were injected. Binding responses on flow cells with immobilized caspase-1
cell. A 1:1 Languir model in BIAevaluation software (GE Healthcare) was used
for fitting the sensograms and the KDvalues were calculated from the ratios
Immunoprecipitation from THP-1 Cell Extracts. THP-1 cells were grown to a
Cells were lysed by Dounce homogenizer in ice-cold buffer (20 mM Hepes-
KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM Na-EDTA, 1 mM Na-EGTA, 0.1
by centrifugation at 500 ? g, 3,000 ? g, and 22,000 ? g for 10 min each.
Aliquots were incubated with or without IgGs overnight at 4 °C and immu-
nocomplexes were recovered by protein-G agarose beads. Presence of pro-
caspase-1 and caspase-1 were visualized by Western blot analysis.
Immunofluoresence Microscopy. THP-1 cells were grown to the density of 5 ?
105cell/ml and differentiated with 0.5 ?M PMA for 3 h and allowed to attach
to no. 11⁄2 glass coverslips overnight. Cells were treated with 1 mM LPS for 2 h
followed by 5 mM ATP for 30 min before fixation and mild detergent perme-
abilization. After blocking with 10% BSA for 1 h, IgGonwas added at 100 ?M
concentration for 1 h. After 3 washes with PBS ? 0.1% Triton X-100, the cells
were stained for 1 h with Alexa Fluor 488 conjugated goat anti-human IgG
antibody (Invitrogen). Cells were washed 3 times and mounted with ProLong
Gold containing DAPI (Invitrogen). Images were recorded on a Nikon 6D
High-Throughput Microscope equipped with a Photometrics Coolsnap HQ2
ACKNOWLEDGMENTS. We thank S. Birtalan, Y. Zhang, B. Li, G. Fuh, J. Scheer,
V. Chiang, and Y. Chen for advice and assistance. We also thank the Protein
Engineering Department and the sequencing, oligonucleotide synthesis, and
fermentation teams at Genentech for generous support, as well as K. Thorn
and the Nikon Imaging Center at University of California, San Francisco, for
help with immunofluorescence microscopy. This work was supported by Na-
tional Institutes of Health Grant 5R01AI070292–02 and the Sandler Family
Foundation gift (to J.A.W.).
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