Materials and Methods
Materials
P-Adnectin (AdP) was provided by Bristol-Myers Squibb. Amicon® Ultra-15
centrifugal filter units (NMWL/MWCO of 30 and 100 kDa) were purchased
from Merck Millipore (Darmstadt, Germany). Citric acid, tribasic sodium
citrate dihydrate, Tris-HCl, Trizma base, sodium hydroxide, L-cysteine,
sodium phosphate (monobasic and dibasic), sodium sulfate, triisopropyl
silane (TIPS), trifluoroacetic acid (TFA), dichloromethane (DCM), were
purchased from Sigma Aldrich (St. Louis, MO/USA). Dimethylformamide
(DMF), piperidine, N-methyl pyrrolidone (NMP), activator
2-(1H -benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU) and methyl-tertiary-butyl ether (MTBE) were
purchased from AGTC Bioproducts (Wilmington, MA). Nova-PEG Rink Amide
Resin was purchased EMD MilliporeSigma (Burlington, MA). All
20-fluorenylmethoxy carbonyl (Fmoc) amino acids were purchased from 21st
Century Biochemicals (Marlborough, MA). Fmoc-Lys-FAM-OH was obtained
from AAT Bioquest (Sunnyvale, CA). 10X SDS PAGE running buffer,
Kaleidoscope Precision-Plus protein gel standard ladder, Bio-Rad AnykD
polyacrylamide gel and SDS-PAGE system were procured from Bio-Rad
Laboratories. Yeast extract was obtained from Amresco, while
Bacto-tryptone, Bacto-agar were obtained from BD.
Single stranded oligonucleotide sequences and gBlock gene fragments
encoding the peptide/linker sequences were purchased from Integrated DNA
Technologies (IDT). Pfu Ultra II Hotstart PCR Master Mix for cloning was
obtained from Agilent Technologies. NEB PhD-12 Phage Display Library
Kit, DH5α variant NEB5α cells, DpnI, BamHI, EcoRI, NdeI and PstI
restriction enzymes were purchased from New England Biolabs (Ipswich,
MA). QiaPrep spin Miniprep kit was ordered from Qiagen (Frederick, MD).
Bio-Rad Laboratories MyCycler was used for carrying out PCR. Solid black
384 well plates (CAT 3575) from Corning were used for fluorescence
polarization assays. BEH C4 RPLC and BEH 200A SEC columns were obtained
from Waters (Milford, MA).
Equipment: Bruker MALDI-Tof II equipment was used for mass spectrometry
of the different protein components used in experiments. Intavis
Multipep RS instrument was utilized for solid phase peptide synthesis. A
Waters Acquity ultra performance liquid chromatography system was
utilized for the UPLC analysis of experimental samples.
Phage Biopanning
One well in a 96 well plate was incubated for 12 hours with the target
AdP in PBS pH 7.4 at 40C. Following physisorption, the
well was blocked with bovine serum albumin (BSA) for 2 hours at
40C. Any excess unbound BSA was washed away using PBS
pH 7.4. A 10 μl aliquot of the NEB Ph.D.™-12 phage display peptide
library containing approximately 1011 plaque forming
units (pfu) was then mixed into 140 μl PBS pH 7.4 in a 0.5 mL centrifuge
tube and added to the well. All steps of incubation, washing and elution
were carried out according to the protocol detailed in the NEB phage
display manual at room temperature (230C). 10 washes
comprising combinations of PBS and PBST (PBS + 0.1% Tween-20) were
performed to remove weakly bound phages from the immobilized AdP.
Following the washes, elution was carried out using 150 μl 0.2 M Glycine
pH 2.2 for 15 minutes on a rotating platform. The eluate was immediately
neutralized by adding 1 M Tris pH 9.1. Titering of the eluate followed
by amplification was carried out according to the NEB phage display
manual. This procedure constitutes a single round of biopanning.
The amplified eluate was diluted approximately to 1011pfu and two additional rounds of biopanning were performed. In the
subsequent biopanning rounds, stringency of washing was increased by
using PBST with a higher concentration of Tween-20 (0.5%). The final
eluate obtained at the end of the third round of biopanning was not
further amplified. The unamplified final eluate was titered and 20
unique distinct phage plaques were picked, amplified and the phage
supernatants were submitted for sequencing. Sequencing was performed by
Genewiz (South Plainfield, NJ) using the -96 gIII sequencing primer 5´-HOCCC TCA TAG TTA GCG TAA CG –3´ and sequences were
analyzed using Expasy Translate
(https://web.expasy.org/translate/). Figure 1 presents a schematic
of the phage panning approach used to identify peptide ligands for AdP.
Peptide Synthesis
Peptides were synthesized with a C-terminal
5(6)-carboxyfluorescein-labeled lysine, by solid phase peptide synthesis
(SPPS) on an Intavis AG Multipep RS (Chicago, IL).
Fluorenylmethoxycarbonyl (Fmoc) chemistry was used to synthesize the
peptides (Chandra et al., 2013). Peptide purity was determined by a
Waters Acquity ultra-performance liquid chromatography (UPLC) system on
a Waters BEH C18 reversed phase liquid chromatography (RPLC) column.
Fluorescence Polarization
Stock solutions of labeled peptides were prepared at a concentration of
500 nM. Protein solutions of a wide range of concentration (0.5 μM-200
μM) were prepared. Typically, 12 protein dilutions were prepared and the
change in fluorescence polarization (FP) signal was recorded. 20 μl of
labeled peptide was incubated with 20 μl of each protein solution in
384-well black plates. Each peptide-protein concentration pair was
analyzed in duplicate. Peptide and protein were incubated with shaking
for 3 hours at room temperature, following which the FP values were
recorded in a Biotek Synergy HT plate reader. Excitation was carried out
using a 485 ± 20 nm excitation filter and emission was controlled by a
520 ± 20 nm filter, 20 nm being the spectral bandwidth of each filter.
Gain and top probe vertical offset were appropriately modified to obtain
analyzable intensity values. Fluorescence polarization values obtained
from the experiment were then fitted to a 4-parameter logistic equation
(Equation 1) to obtain binding affinity (‘C’ parameter) (Gadagkar &
Call, 2015). Normalized FP change was calculated as (FP –
FP0)/FP0, where the FP is the
fluorescence polarization value at a given concentration while
FP0 is the base polarization value (at zero target
concentration).
\begin{equation}
Y=\ \frac{(A-D)}{\left(1+\left(\frac{X}{C}\right)^{B}\right)}+D\nonumber \\
\end{equation}Equation 1: 4 parameter logistic curve equation used to fit binding
curve data. A = response at zero analyte concentration, B = slope
factor, C = inflection point, D = response at infinite analyte
concentration
Peptide-ELP Cloning, Expression and
Purification
Site-directed mutagenesis using Pfu Ultra Hotstart 2x PCR Master Mix,
was used for the creation of peptide-ELP constructs. For the P10 lead
peptide, two sets of completely overlapping forward and reverse primers
were designed to sequentially clone in one half of the peptide first,
followed by the remaining half. The PCR reaction recipe comprised: 1 μl
template DNA (~50-100 ng/μl), 1 μl of each primer (to a
final concentration of 0.5 μM), 25 μl of PfuUltra II Hotstart PCR Master
Mix, 2.5 μl of DMSO (if required to reduce the melting temperature of
primers) and the remaining volume of ultrapure water to make up the
reaction volume to 50 μl. Denaturation was carried out at
950C for 30 seconds, followed by an annealing step for
20 seconds. The annealing temperature was set depending on the melting
temperatures (Tm) of the primers. For primers with
Tm >720C, the annealing
temperature was kept between 68 and 720C, whereas for
primers with lower Tm values, annealing temperatures
between 58-620C were used. Following the PCR, DpnI
digestion was conducted to degrade parental template DNA for 1 hour at
370C. The resulting DNA was transformed into NEB5α
cells using thermal shock. Transformant colonies were picked from the 50
μg/mL Kanamycin plates and grown overnight in LB media. DNA was
extracted from the grown cultures by minipreparation using the QIAPrep
Spin Miniprep Kit. Sequences were analyzed for correctness using Expasy
Translate.
The correctly identified DNA sequences were transformed into BL21DE3.
Transformant colonies were picked and grown in 5 mL LB media cultures in
the presence of 50 μg/mL Kanamycin before addition to 250 mL Terrific
Broth (TB) containing 50 μg/mL Kanamycin. Two routes of expression were
tested. The first involved induction with IPTG at OD600of 0.8-1.0 (1 mM final concentration) and the second was performed in
the absence of IPTG. Both routes delivered significantly high expression
yields. In the case of IPTG induction, the 250 mL culture was grown at
370C for 4-5 hours until it approached an
OD600 value of 0.8-1.0. IPTG was added to a final
concentration of 1 mM and the temperature was reduced to
220C to allow for protein expression to occur. For the
expression involving no IPTG addition, the culture was grown for 16-20
hours at a constant temperature of 370C. The culture
was then centrifuged at 4000g for 20 minutes and the cells were
pelleted. The cell pellet was re-suspended in 10 mL 1X PBS with protease
inhibitor cocktail and subjected to sonication (in pulses of 5 seconds
ON, 5 seconds OFF) for 10 minutes. The lysed cells were centrifuged for
15 minutes at 15,000g and the pellet was discarded. Inverse transition
cycling, used in previous ELP purification protocols (Sheth et al.,
2013) was also employed here to purify the ELP constructs. The ELP
containing supernatant was isolated and 4 mL of 1 M
Na2SO4 was added to precipitate the ELP.
Centrifugation was performed at 370C at 15,000g for 15
minutes to pellet the precipitated ELP and the impure supernatant was
discarded. The pellet containing ELP was then resolubilized in cold PBS
(40C) and the solution was centrifuged at
40C at 15,000g for 15 minutes to remove any insoluble
impurities. This cycle was repeated at least once more in order to
obtain considerably pure ELP construct. Purity of the ELP construct was
determined by C4-RP-UPLC analysis and used for further
experiments.
Pure Component AdP Precipitation
Experiments
A schematic of the ELP-peptide affinity precipitation process is
presented in Figure SI-1. Preliminary binding screening experiments and
elution tests were performed in conical 1.5 mL Eppendorf tubes. Elution
screening experiments involving mobile phase modifiers were performed
using ‘V’-bottomed 96-well plates, in order to increase the throughput
of the experiments. In all cases, concentration of AdP used was 40 μM (1
mg/mL). Also, in all cases, 50 μL of 40 μM AdP was mixed with 50 μL of
peptide-ELP construct (at various molar ratios) and incubated for 20
min. 50 μL of 1 M Na2SO4 (final
Na2SO4 concentration of 0.33 M) was then
added to the mixture to precipitate the peptide-ELP-AdP complex.
Centrifugation was carried out at 3200g for plate-based experiments
(10000g for tube-based experiments) for 15 minutes to ensure complete
precipitation and pelleting of the complex. 145 μL of the supernatant
was cleared and recorded as ‘S1’ or the first supernatant, which
accounted for the unbound AdP. Experiments for the quantification of
only the pulldown of AdP into the precipitate form were stopped at this
point and the amount of AdP in the supernatant was used to measure
percentage pulled down. At this point, absence of any AdP in the
supernatant would indicate complete pulldown. In this case, the
precipitate (denoted as P1) was re-solubilized in PBS pH 7.4 and
subjected to analysis.
In the case of elution screening experiments, the precipitated pellet
was then re-solubilized in 100 μL of various elution buffers tested.
Following re-solubilization of precipitate, 50 μL of 1 M
Na2SO4 prepared in the elution buffer
was added in order to precipitate the peptide-ELP construct. At this
point, complete elution would result in precipitation of only the
peptide-ELP, leaving only the AdP in the supernatant (S2, or the second
supernatant). However, incomplete elution would result in the
precipitation of peptide-ELP as well as any undissociated
peptide-ELP-AdP complex upon Na2SO4addition. Centrifugation was performed at 3200g for plate-based
experiments (10000g for tube-based experiments) for 15 minutes at
370C to pellet the precipitate. 145 μL of the
supernatant S2 was cleared and the pellet, or second precipitate
(denoted as P2), was re-solubilized in 150 μL PBS at pH 7.4. The S1, S2
and P2 fractions were analyzed for each experiment using a reversed
phase UPLC assay.
Purification of AdP from Crude
Mixtures
.
Pure AdP was spiked into E. coli lysate to a final concentration
of 40 μM. The AdP-spiked crude mixture was then adjusted to pH 4 which
was was identified as the optimal binding condition. Pprecipitate formed
during this pH adjust (containing process related impurities) was
removed by centrifugation and the supernatant was collected. 50 μL of
the peptide-ELP construct was then added to 50 μL of the supernatant and
incubated for 20 minutes at 230C. 50 μL of 1 M
Na2SO4 in binding buffer was added to
the AdP-peptide-ELP mixture and centrifuged for 15 min at 10000g. The
supernatant S1 was discarded and the pellet was washed three times with
150 μL 0.33 M Na2SO4 in binding buffer.
The precipitate was then re-solubilized and eluted in 200 μL of elution
buffer (50 mM Tris, pH 8.5 with 500 mM arginine). 100 μL of 1 M
Na2SO4 in elution buffer was added to
the solution and centrifuged at 10000g for 15 min to pellet the
precipitate containing only dissociated peptide-ELP construct. The
purified AdP supernatant S2 was removed and all the fractions of the
experiment were evaluated for extent of purification and recovery using
UPLC.
C4 Reversed Phase UPLC
Analysis
An Acquity UPLC Protein BEH C4 column was used for the analysis of all
fractions obtained from the binding, precipitation and elution
experiments. Linear gradients were carried out at 0.4 ml/min using two
buffers: 5 % acetonitrile, 0.1 % trifluoroacetic acid in water (Buffer
A) and 95% acetonitrile, 0.1% trifluoroacetic acid in water (Buffer
B); all % in v/v. The gradient consisted of a 0-30% B gradient in 1.2
column volumes, followed by a 30-70% B gradient in 3 column volumes and
a 70-100% B gradient in 1.2 column volumes. A regeneration using 100%
B was then performed for 1.6 column volumes, followed by a column
re-equilibration step at 0% B for 1.6 column volumes. The column
effluent was monitored at 280 nM to quantify the amounts of AdP and
peptide-ELP present in the fractions.
SDS PAGE Analysis
Samples were analyzed for purity using SDS PAGE. 25 μL of each sample
was mixed with 25 μL Laemmli sample buffer and boiled at
950C for 5 minutes. A protein ladder was injected in
the first well of a Bio-Rad AnyKd polyacrylamide gel. 25 μL of prepared
samples were injected into the wells and the gel was run in a Bio-Rad
mini-PROTEAN Tetra Cell system in the presence of running buffer (1X
Tris/Glycine/SDS buffer). The gels were washed with water and then
stained using Coomassie-Blue for 45 minutes, followed by de-staining
using deionized water.