From the Affymax Research Institute, Santa Clara, California 95051
Received for publication, December 28, 2000
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ABSTRACT |
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Peptide libraries generated using phage display
have been widely applied to proteolytic enzymes for substrate selection
and optimization, but the reaction kinetics between the enzyme and substrate phage are not well understood. Using a quantitative ELISA assay to monitor the disappearance of substrate, we have been able to follow the course of reaction between stromelysin, a
metalloprotease, and its substrate phage. We found that under the
proteolytic conditions where the enzyme was present in nanomolar concentration or higher, in excess over the substrate, the proteolysis of substrate phage was a single exponential event and the observed rate
linear with respect to enzyme concentration. The enzyme concentration dependence could be described by pseudo first-order kinetic equations. Our data suggest that substrate binding is slow relative to the subsequent hydrolysis step, implying that the phage display selection process enriches clones that have high binding affinity to the protease, and the selection may not discriminate those of different chemical reactivity toward the enzyme. Considering that multiple substrate molecules may be present on a single phage particle, we
regard the substrate phage reaction kinetic model as empirical. The validity of the model was ascertained when we successfully applied it to determine the binding affinity of a competitive inhibitor
of stromelysin.
Since the first reports of the display of randomized peptide
libraries on the surface of filamentous bacteriophages (1-3), phage
display has become a widely adopted approach for the mapping of
antibody epitopes and macromolecule-binding peptides (4-6), the
selection of peptide agonists or antagonists against potential drug
targets (7, 8), and the selection and optimization of sequence
determinants that facilitate protein folding and stability (9-12). An
important application of phage display of peptide libraries is in the
selection and optimization of substrate for proteases and protein
kinases (13-15). Traditionally, the sequence of a peptide substrate
for protease is deduced from the natural cleavage sites of the in
vivo polypeptide substrates if known. There is usually conservation of amino acid residues at the site of cleavage and in the
flanking sequences. However, it has been shown that the cleavage
sequences from the natural substrates are not always optimal for the
enzymes when the enzymatic activities are studied in vitro
with synthetic peptides (16-18). Peptide libraries generated through
phage display have been instrumental in optimizing substrates and
mapping the sequence preferences at both P and P' sites (13, 16-19).
The insertion of randomized sequences into protein pIII (product of
gene III) of filamentous phage is the most common approach for making
phage display libraries. There are up to five copies of pIII present on
the surface of the phage when gene III is expressed (20). Two
strategies, monovalent and multivalent display, have been reported for
protease substrate phage selections (13, 19). In either case, gene III
is modified such that a randomized sequence is linked to the N terminus
of pIII and an affinity tag, which may be a protein or peptide epitope
or a histidine tag sequence, is linked N-terminal to the random
sequence (13, 19, 21, 22). The phage particle loses the epitope/tag if
a peptide in the library serves as a substrate for and is cleaved by
the protease of interest. Unreacted phage particles have the intact
epitope/tag and can be trapped preferentially by affinity binding. The
phage display approach has been successfully applied to a number of different endopeptidases (13, 16-19, 23, 24). The procedures involve
iterative rounds of selection and enrichment aiming at enriching
reactive phage particles containing sequences cleavable by the
protease. The sequences found in the reactive clones are then
transferred from the phage context and placed within a synthetic peptide or a chimeric protein for further characterization.
The assumption has been that the peptides, which were selected from the
substrate phage display, would have better
kcat/Km values over those not
selected (13). However under normal selection conditions, the enzymes
are present at nanomolar concentrations or higher whereas the phage
particles at Phage is an unusual substrate for enzymes in that the phage particles
present multiple copies of the substrate (the recombinant pIII) at one
tip of a long cylinder (20), creating microscopic heterogeneity. It is
therefore not clear whether the conventional single-turnover kinetic
considerations would still apply to the reaction of substrate phage.
However, a good understanding of the reaction kinetics is fundamentally
important and necessary for rational experimental designs. The present
study sets out to define the kinetic properties of the reaction between
protease and substrate phage. Using the metalloprotease stromelysin as a model, we examine in detail the kinetics of the reaction between the
enzyme and the isolated substrate phage clones. We put forth a kinetic
model and further validate it using a competitive inhibitor of the
enzyme. Our kinetic model will provide more rational considerations to
future phage display experimental designs, which up to now have been
highly empirical.
Materials and Buffers--
Streptavidin and bovine serum albumin
(BSA)1 were purchased from
Sigma and ECL blotting detection reagent and HRP/anti-M13 monoclonal
conjugate from Amersham Pharmacia Biotech (Piscataway, NJ). The
biotinylated mouse monoclonal antibody 179 (mAB179) was prepared in
house. The phage clones were maintained and amplified in
Escherichia coli K91 cells (19). Recombinant stromelysin was
prepared as reported previously (19), and its concentration was
calculated based on the total protein concentration of the protein
preparation. ELISA wash solution was 0.05% Tween 20 in PBS and phage
assay stop solution 0.1% BSA and 5 mM EDTA in PBS.
Phage Preparation and Phage ELISA Assay
Procedures--
Typically the E. coli cells harboring the
phage were cultured in 1-liter quantities, overnight at 37 °C. The
phage particles were precipitated from the supernatant following a
general procedure described previously (19). Phage preparations from
1-liter cultures were typically reduced to a volume of 1 ml in 25 mM Tris, pH 7.4, 20% glycerol, and 75 mM NaCl
and then titered.
Plates used for ELISA assays were Microlite 2, white 96-well flatbottom
plates from Dynex Technologies (Chantilly, VA). They were first coated
with Streptavidin at 10 µg/ml in 10 mM PBS, pH 7.4 using
200 µl/well and incubated for 1 h at 37 °C. The next coating
was done with biotinylated mAB179 at 2 µg/ml, 200 µl/well in PBS
for 1 h at 37 °C. Finally, a solution of 1% BSA and 10% sucrose in PBS was added at 250 µl/well, and the plates incubated for
1 h at 37 °C. The plates were washed five times with ELISA wash
solution between each coating. After the last coating, plates were
aspirated and allowed to air dry.
For each ELISA assay, 200 µl of phage-containing samples were added
per well, and the plate was put on a titer plate shaker at room
temperature for 1 h, after which the plates were washed five times
with the wash solution. HRP/anti-M13 monoclonal conjugate, diluted
40,000 times in 0.1% BSA in PBS at 200 µl/well, was added, and the
plates were put on the plate shaker for 30 min at room temperature. The
dilution of the HRP/Anti-M13 was optimized for each lot. Again, the
plates were washed five times in wash solution. Finally, ECL blotting
detection reagent was added at 200 µl/well (no incubation) and the
chemiluminescence was read in Packard Topcount (Packard Instrument,
Meriden, CT) in single photon count mode at 0.02 min/well.
The plate coating and the ELISA procedures were also automated. Upon
completion of the transfer of the digest reaction to the ELISA plates
by the Tecan (see below), a Sagian robotic arm transferred the plates
to a Scitec Heidolph Titramax 100 plate shaker for the time indicated
above. The Sagian robotic arm would then transfer the plates to an SLT
96 plate washer. All other reagents used for the ELISA were dispensed
by Cavro syringe pumps attached to eight-pronged manifolds. The robotic
arm then transferred the plates to a dedicated Packard Topcount
adjacent to the robotic table. All robotics were controlled by a
Pentium PC.
Quantitative Phage ELISA Assay--
The standard curves of each
preparation were determined by serial diluting the phage in phage assay
stop solution and running the ELISA without protease. The starting
concentration for phage in the protease digest was determined from the
standard curve by choosing the concentration coinciding with the
uppermost part of the "useful range" (see Fig. 2). 200 µl of the
stopped digest reaction (see below) were used in the ELISA assay. As a
zero time, we included a tube of only phage and no enzyme. Every ELISA
plate included a standard curve of phage and zero time points for
quantification of every time point.
Procedures and Kinetics of Proteolytic Digest of Substrate
Phage--
Phage digests for stromelysin were typically done in
150-µl reaction volumes of 20 mM HEPES, pH 7, 5 mM CaCl2, 0.02% Brij 35 containing stromelysin
at concentrations between 5 nM and 1.2 µM.
The reaction tubes containing the lower concentrations of stromelysin
were put on a slower time course, and the tubes containing higher
concentrations were put on a faster time course to sample enough points
for the steepest part of the decay curve (see Fig. 3). The digest
reaction was initiated by adding substrate phage at a concentration ten
times higher than that determined above so that upon stopping the
digest by dilution, the signal intensity would lie on the uppermost
part of the useful portion of the standard curve (see Fig. 2). 15-µl
time points were removed from the digest reaction and diluted 20 times
in stopping solution to inactivate stromelysin. 200 µl of this
solution were then removed and subjected to the quantitative phage
ELISA assay to determine the concentration of the remaining substrate
phage. For each stopped reaction, the ELISA assay was performed in
triplicate and the averaged results graphed. The enzyme
concentration-dependent phage digest experiments were
performed over a course of several days, and data collected on
different days were reported on the same graph.
When stromelysin inhibitor studies were performed, an enzyme
concentration was chosen that would produce a well-defined digest curve
reaching 100% digested within 20 min. Various concentrations of
inhibitor were added to the reaction tubes and allowed to incubate with
stromelysin for ~5 min before initiating the digest with a
concentrated stock of phage. All linear and nonlinear regression analyses were performed using KaleidaGraph 3.09 by Synergy Software (Reading, PA).
Eventually, all of the above steps were programmed and performed with a
robotic system from Scitec Inc. (Ashland, MA). The 1.2-ml digest tubes
were held in a heating block that was kept at 25 °C by a circulating
water bath. A Tecan liquid handler removed time points from the digest
reactions and transferred them to a deep-well plate containing the stop
solution. Sample was mixed three times and then waited until the next
time point. When all the time points were completed, the Tecan
transferred the stopped digest reaction from the deep-well plates into
the ELISA plates. At this point, the ELISA assays were run either
manually or robotically.
Quantitative Determination of Substrate Phage--
Fig.
1 shows the substrate phage clones used
in the present study. The presence of the mAB179 epitope allowed the
phage particles to be captured in wells of a microtiter plate that had
been precoated with streptavidin followed by biotinylated mAB179. The
captured phage was then detected using HRP-conjugated anti-M13 antibody and a chemiluminescent peroxidase substrate. When the activity of HRP
was developed, the chemiluminescence signals showed a sigmoidal function with respect to phage titer on a logarithmic scale (Fig. 2A). The signals were highly
specific to the phage clones containing the mAB179 epitope. The
parental phage clone (fdTET) containing the wild-type pIII at
1010 cfu/ml did not cross-react at all (Fig.
2A). The change of the chemiluminescence signal was most
sensitive to the change in the phage concentration on the steepest
portion of the curve. This part of the curve thus served as a standard
curve against which a sample of unknown phage titer could be quantified
(Fig. 2B).
All phage clones investigated in the present study were identical
except in the protease substrate sequence (Fig. 1). We found that each
clone showed noticeable but only slight variations in the shape of the
standard curve when a complete titration curve was constructed for each
of the clones (results not shown). To maintain the accuracy, a standard
curve was constructed with the same clone that was being tested, and
the curve was repeated on each of the test plates to avoid
plate-to-plate variation.
Reaction Kinetics of Protease with Substrate Phage--
The time
course for the proteolysis of clone 10629 by stromelysin is shown in
Fig. 3. 3 × 1011 cfu/ml
of 10629 was incubated with stromelysin at 25 °C. A sample was
removed at each time point, and the reaction was terminated by the
addition of EDTA to 5 mM final concentration. The amount of
substrate phage particles remaining in the sample was determined by the
quantitative phage ELISA assay as described under "Experimental Procedures." The time course was best fitted to a single-exponential equation using nonlinear regression (Equation 1),
Pseudo First-Order Kinetic Model of Reaction between Enzyme and
Substrate Phage--
Proteolysis reactions of substrate phage are
normally performed with solutions containing
We determined the single-exponential rate constants
(kobs) for five different phage clones (Fig. 1)
at concentrations of stromelysin ranging from 5-1200 nM
(Fig. 4). The reactivity of the clones is
widely different; however, without exception, the
kobs appears linear to the enzyme concentration
throughout the range. Our data clearly agree with Equation 2,
suggesting that the slopes of the linear plots should reflect
k1, the second-order rate constant for substrate
binding to enzyme.
Recombinant proteins displayed on the tip of bacteriophage particles
are not ordinary substrates for enzymes. Phage particles are long
cylinders of about 7 nm in diameter and 1-2 µm in length, and each
can have five copies of protein pIII (20). Because pIII is present only
on one end of the cylinder, there is microscopic heterogeneity in
substrate distribution. Interestingly, our data (Figs. 3 and 4)
apparently agree with the conventional single-turnover kinetic
equations and indicate nothing out of the ordinary on the macroscopic
level for the enzymatic reactions involving substrates displayed in
multivalencies. We thus conclude that the reaction between an enzyme
and its substrate phage obeys the rules of pseudo first-order kinetics.
However, we propose that the pseudo first-order kinetic model for
substrate phage be regarded as empirical, considering that differences
do exist microscopically between homogeneous substrate entities (such
as synthetic peptides or purified recombinant proteins) and the
recombinant protein substrate generated through phage display.
Application of the Kinetic Model to Inhibitor Study--
We
further tested the pseudo first-order kinetic model with clone 10519 by
using it to investigate the binding affinity of a competitive inhibitor
of stromelysin. A diketopiperazine inhibitor was isolated from a
combinatorial synthetic compound library and found to have an
IC50 of 9.6 µM against
stromelysin.3 In the presence
of a competitive inhibitor, the reaction scheme illustrated in Reaction
1 becomes the pathway delineated in Reaction 3.
We determined the kobs of the proteolysis of
clone 10519 in the presence of varying amounts of the inhibitor. The
results, shown in Fig. 5A,
indicate that the presence of the inhibitor resulted in a decrease in
the rate of proteolysis. Fig. 5B shows the complete plot of
the kobs values as a function of inhibitor concentration and the least-squares fit by nonlinear regression to
Equation 5. The fit yielded a Ki of 3.8 ± 2.6 µM, which within experimental error is in good agreement
with that predicted from the IC50 value.3
We have shown that the reaction of substrate phage with enzyme is
a single-turnover event and can be defined by equations of pseudo
first-order kinetics. The single exponential rate of product formation
reflects the apparent bimolecular binding rate between the enzyme and
the substrate.
Several protocols have been successfully applied to the selection of
protease substrate phage through screening phage display peptide
libraries. The phage particles in the library are either incubated
directly with the enzyme in solution (19) or pre-bound to a solid
support followed by incubation with the enzyme (13, 21). Based on our
finding that the proteolysis reaction is single exponential, the
selection processes should enrich phage clones that have shorter
half-lives from the proteolysis. A shorter half-life results from a
faster rate of substrate association with enzyme, i.e.
larger kobs. If it were possible to perform
steady-state kinetic experiments with substrate phage, it would imply
that substrates with larger kobs would have a
smaller Michaelis constant Km and a larger
kcat too, if the turnover number was somewhat
limited by the binding step. Because it is not feasible experimentally
to achieve the conditions of steady-state kinetics with substrate
phage, synthetic peptides with the sequence encompassing the entire
variable region of the substrate phage have often been used to further
characterize the substrates identified in phage display experiments.
The second-order rate constants calculated from the linear plots in our
single-turnover studies, shown in Fig. 4, are summarized in Table
I along with the steady-state kinetic
parameters measured previously for the corresponding synthetic peptides
(19). The second order rate constants vary over 100-fold for the five
phage clones tested whereas the
kcat/Km values differ by
30-fold for the synthetic peptide substrates. It appears that
Km, and kcat to some extent,
contribute to the increase in the enzyme catalytic efficiency
(kcat/Km) of the synthetic
peptides. It has been shown in several studies that synthetic peptides, derived from substrate phage clones selected under the same condition, could have very different Km and
kcat values (16-18), and furthermore, there
were even cases where peptide sequences derived from reactive phage
clones failed to show significant reactivity when incorporated into
synthetic peptide (19). It can be concluded that overall there is no
clear distinction as to which one of the steady-state kinetic
parameters is the prevailing contributor to the improvement of
catalytic efficiency. Furthermore, there may be no direct correlation
between the reactivity of phage clones and synthetic peptides. This may
be a consequence of the fact that phage and peptide are two unrelated
substrate systems.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1012 particles/ml, which is equivalent to
1.66 pM, or 8.3 pM if taken into account that
up to five copies of pIII may be expressed on the surface of a given
phage particle (20). In any case, the enzyme is in excess over the
substrate, indicating a single-turnover reaction to which it is invalid
to apply the equations for steady-state kinetics.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (11K):
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Fig. 1.
Recombinant pIII sequences of substrate phage
clones for stromelysin. The clones, created earlier (19), are
identical except in the variable region where the proteolytic cleavage
occurs. The arrow indicates the start of the native pIII
protein, and the underlined sequence is the epitope of
mAB179.
View larger version (16K):
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Fig. 2.
Quantitative ELISA assay curves.
A, the chemiluminescence readouts in CPS are plotted as a
function of the phage titer on a logarithmic scale for clone 10519 ( ) and the control fdTET (
), and the line is the
sigmoidal fit to the data using nonlinear regression. B, the
useful portion of the curve serves as a standard curve for quantifying
the amount of intact clone 10519 remaining in reaction mixtures.
View larger version (17K):
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Fig. 3.
Time course of proteolysis of clone 10629 by
stromelysin. The starting substrate phage titer was 3 × 1011 cfu/ml, and the enzyme concentrations were 40 ( ),
80 (
), and 120 nM (
). The lines are the
fit to Eq. 1.
and the rate of substrate phage disappearance
(kobs) was calculated. [S]0 is the
starting substrate concentration. Because the phage ELISA assay
determines the total substrate concentration (both enzyme-bound and
unbound, see Reactions 1 and 2), the rate for substrate phage
disappearance is also the measurement of the rate for product
formation. That the product formation from substrate phage follows the
single exponential kinetics does not seem to be limited to stromelysin,
which is a metalloprotease. We have made similar observations with
serine proteases including viral and bacterial
enzymes.2
(Eq. 1)
1012 cfu/ml
phage (13, 19), which corresponds to an effective concentration of the
recombinant pIII up to 1.66 or 8.3 pM in monovalent or
multivalent display, respectively. On the other hand, the
concentrations of protease used in phage display experiments are
generally in nanomolar to micromolar range, which is much greater than
a substrate concentration of 8.3 pM. Therefore, the reaction of the enzyme with substrate phage can be regarded as a pseudo
first-order process with respect to the substrate (see Reactions
1 and 2). The general reaction scheme for protease-catalyzed substrate
hydrolysis is shown in Reaction 1,
where P and P' are hydrolyzed products of S, and P may be
covalently linked to the enzyme in the case of serine or cysteine proteases. When [E]0 >> [S]0, the
second-order reaction can be reduced to a pseudo first-order process as
is shown in Reaction 2.
Our experimental data showed clearly that the product formation,
reflected by substrate disappearance, was a single-exponential process
(Fig. 3), which further suggests that either the substrate binding or
the hydrolysis step is fast. For the pseudo first-order scheme shown in
Reaction 2, the apparent single-exponential rate constant for product
formation is shown in Equation 2,
if the substrate hydrolysis is fast relative to the binding step;
that is, there is a linear function between kobs
and enzyme concentration. If the binding step is fast, the apparent
rate constant for the product formation is shown in Equation 3,
(Eq. 2)
where Kd1 = k
(Eq. 3)
1/k1.
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Fig. 4.
Dependence of the observed first-order rate
constants of proteolysis on enzyme concentration. The observed
single-exponential rate constants (kobs) were
determined at various enzyme concentrations for each of the five
substrate phage clones: 10519 ( ), 10629 (
), 5-2 (
), Good
(
), and 10517 (
). The kobs values are
plotted as a function of stromelysin concentration, and the lines
are the linear curve fit to the data. The fit yielded
k1, the second-order rate constant for substrate
association with enzyme, and k
1, the
dissociation rate constant. k1 values are listed
in Table I whereas k
1 are 0.011, 0.016, 0.008, 0.052, and 0.012 min
1 for clones 10517, Good, 5-2,
10629, and 10519, respectively.
In the presence of excess inhibitor, the free enzyme concentration
is shown by Equation 4.
Under our assay conditions, the substrate phage titer was on the
order of 8 × 109 cfu/ml and the enzyme in the
nanomolar range. The presence of inhibitor at 10 times the dissociation
constant Ki does not change the pseudo first-order
assumption. Thus, Equation 2 can be modified to account for the
inhibitor as is shown in Equation 5,
(Eq. 4)
and the Ki can be determined by varying the
inhibitor concentration.
(Eq. 5)
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Fig. 5.
Effect of a competitive inhibitor on the
proteolysis of substrate phage. A, the time course of
the proteolysis of clone 10519 in the presence of 0 ( ), 2.5 (
),
and 15.6 µM (
) of the diketopiperazine inhibitor of
stromelysin. The enzyme concentration was 40 nM.
B, the complete data set plotted as a function of the
inhibitor concentration. The line is the least-squares fit
of the data to Eq. 5, yielding Ki = 3.8 ± 2.6 µM, k1 = (3.5 ± 0.7) × 104 M
1 s
1 and
k
1 = 0.021 ± 0.018 min
1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Comparison of substrate phage reactivity with that of synthetic peptide
substrates
We recognize that recombinant proteins expressed on the tip of a phage particle and synthetic peptides are different molecular entities and as substrates, the substrate phage obeys single-turnover kinetics whereas the synthetic peptides are usually assayed under multiple-turnover conditions obeying steady-state kinetics. This may create difficulties in extrapolating the phage display results to the synthetic peptides. Another consideration for why it is difficult to extrapolate may lie in plausible protein-protein interactions between the enzyme and the bulk of the recombinant pIII protein outside of the substrate region. Several strategies have been employed in designing the flanking sequences for the incorporation of randomized peptides into pIII in the hope of minimizing the effect of the pIII polypeptide outside of the random region (13, 19). It is possible, however, that peptides of certain sequences in the random region could still promote favorable protein-protein interactions between the enzyme and the recombinant pIII, and as a result these clones would be preferentially selected. They may behave poorly as substrate once the substrate sequence is separated from the pIII protein and made into synthetic peptides.
The substrate phage selection procedures rely on capturing and removing unreacted clones from the library, in the hope of enriching reactive ones following each round of selection. That the course of reaction between enzyme and substrate phage is single-exponential (Fig. 3) is thus a key observation. It implies that strong and weak substrates are differentiated by their half-lives of proteolytic cleavage, suggesting that time of incubation and the amount of enzyme are critical factors in designing substrate phage selection experiments. Therefore, few substrates would be found if the selection conditions were too stringent; that is, if the incubation time were too short or the enzyme concentration too low. On the other hand, it would offer little discrimination among good substrates if the conditions were too relaxed.
Stringent conditions are preferred if the population of potential
substrates in the library is relatively large. This may apply to
enzymes that can tolerate a number of amino acid sequence variations in
the substrate. For instance, it has been discovered through practice
that stringent conditions were key to the success of substrate phage
display experiments with protein tyrosine kinases (14, 15, 25). In
general, the selection conditions should be set close to the relaxed
scenario when the reactivity of the substrate phage is unknown, and
especially when the population of potential substrates in the library
is small. This is particularly important for enzymes such as certain
proteases that are highly specific in sequence requirements.
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ACKNOWLEDGEMENT |
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We thank Bruce Mortenson for supplying biotinylated mAB179.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Signature BioScience, Inc., 21124 Cabot Blvd.,
Hayward, CA 94545.
§ Present address: Zyomyx, Inc., 3911 Trust Way, Hayward, CA 94545.
¶ Present address: Hoffmann-La Roche Inc., 340 Kingsland St., Nutley, NJ 07110.
To whom correspondence should be addressed: 3410 Central
Expressway, Santa Clara, CA 95051. Tel.: 408-522-5689; and Fax:
408-481-0522; E-mail: danying_cai@affymax.com.
Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.M011772200
2 N. A. Sharkov and D. Cai, unpublished results.
3 L. Shi, D. Harris, M. Navre, D. Tien, A. K. Szardenings, and D. A. Campbell, unpublished results. The IC50 value was determined using a thiopeptide substrate whose concentration was estimated to be below the Km of the substrate. For a competitive inhibitor, the IC50 value is always higher than the Ki value.
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ABBREVIATIONS |
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The abbreviations used are: BSA, bovine serum albumin; cfu, colony-forming unit; CPS, counts per second; HRP, horseradish peroxidase; mAB179, mouse monoclonal antibody 179; ELISA, enzyme-linked immunosorbent assay.
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