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

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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.