SH2 domain production
SH2 domains were produced as previously described [19]. Briefly sequences encoded in kanamycin-resistant pET28 SacBAPvectors were purchased from the Pawson Lab (Samuel Lunenfeld Research Institute, Canada) and a biotin acceptor peptide (BAP) sequence was cloned into the vectors to give an N-terminal BAP-Histag-SH2 domain sequence. For production in RosettaTM2 (DE3) cells (Novagen, Merk Millipore), overnight starter cultures were grown at 37°C, 230 rpm in TB medium supplemented with kanamycin (50 µg/ml), chloramphenicol (34 µg/ml), and 1% glucose. These were used to inoculate 3 ml cultures of TB kanamycin that were grown at 37°C, 230 rpm until OD600 reached ca. 1.5 and temperature was reduced to 18°C for 1 h before addition of 0.5 mM IPTG and cultures were grown overnight at 18°C, 230 rpm. His-tagged SH2 proteins were purified from clarified culture lysates on a KingFisher™ Flex robotic platform (ThermoFisher) using His Mag Sepharose Ni beads (GE Healthcare), washed (50 mM NaH2PO4; 500 mM NaCl; 20 mM imidazole; pH 7.4) and eluted in 130 µl elution buffer (50 mM NaH2PO4; 500 mM NaCl; 300 mM imidazole; 10% glycerol; pH 7.4). The elution buffer also contained 1 mM TCEP. Samples were flash frozen in liquid nitrogen and stored in aliquots at -80°C
Phage display and phage ELISA
Phage display was completed over four panning rounds, as described previously [19]. Streptavidin-coated wells were used for the first panning round, followed by Streptavidin-coated magnetic beads (Dynabeads®; Life Technologies) and NeutrAvidin-coated wells in the final panning round. For competitive pans, an additional incubation of target-bound phage with 2.5 µg of non-biotinylated target was performed for 24 hours at room temp before elution. Phage ELISAs were conducted as described previously [19], and positive clones sent for sequencing.
Affimer Production
Affimer sequences were cloned into pET11a using the NheI and NotI sites. SH2-binding Affimers were produced in BL21 STAR™ (DE3) E. coli (C601003, Life Technologies, Invitrogen) and affinity purified using Ni-NTA resin as previously described [19]. For HA-tagged Affimers, Affimer sequences were subcloned into kanamycin-resistant pET-lectra vectors with C-terminal HA, 8xHis-tag sequences and produced in BL21 Star™ (DE3) E.coli cells in 24 well plates. Briefly, 200 µl starter cultures were grown at 37°C, 1050 rpm in a 96-well plate for 6–8 h in LB broth kanamycin (50 µg/ml) + 1% glucose. Cultures were used to inoculate 3ml of LB broth kanamycin in round bottom 24-well plates and grown at 37°C, 1050 rpm until OD600 reached ca. 0.8. Protein expression was induced with 0.5 mM IPTG and cultures were left overnight at 22°C, 1050 rpm. Affimer proteins were purified from clarified lysates using His Mag Sepharose™ Ni beads on a KingFisher Flex™ robotic platform, as for SH2-domains with the exclusion of TCEP from the elution buffer. Samples were flash frozen in liquid nitrogen and stored in aliquots at -80°C.
Microarray
Protein microarrays were conducted using HA-tagged Affimer reagents and BAP-tagged SH2 domain proteins. SH2 domain protein samples were diluted to 70 µM in PBS containing 20% glycerol and 10 µl samples added to wells in a 384-well microarray plate (Genetix). Proteins were spotted onto the surface of streptavidin-coated 3D-functionalized glass slides (PolyAn), using an ArrayJet Marathon™ non-contact printer. The system buffer contained 47% glycerol, 0.06% Triton™ X-100 (Sigma-Aldrich),0.04% ProClin™ 200 (Sigma-Aldrich) in ddH2O. Each protein spot consisted of 100 ρl solution, with a typical spot size of 200 µm. Proteins were left to dry onto the surface overnight, in a controlled environment of 18–19°C and 50–55% humidity (using the ArrayJet JetMosphere™ system). Slides were scanned at 532 nm using a GenePix® 4300A scanner (Molecular Devices) to visualise and analyse the printed protein spots for any drying artefacts. Slides were incubated with Blocking Buffer 1 (0.1 M Tris-HCl; 50 mM ethanolamine; 0.05% Tween-20, pH 9.0; 140 µl/well) for 15 min at room temperature. Wells were washed x3 with PBST and blocked additionally with Blocking Buffer 2 (2X Casein Blocking Buffer (Sigma-Aldrich); 0.1 M Tris-HCl, pH 8.5; 140 µl/well) for 30 min at room temperature. Arrays were then incubated with 5 µg/ml Affimer in Blocking Buffer 2 (70 µl/well) for 1 h at room temperature, followed by 3x PBST washes. Bound Affimer was detected using an anti-HA-tag AlexaFluorTM 647 conjugated antibody (1:1,000; Thermo Fisher 26183-A647 diluted in Blocking Buffer 2 (70 µl/well), for 1 h at room temperature in the dark. Negative control miniarrays were included on each slide; these controls were incubated with Blocking Buffer 2 and HA-tag antibody only. Slides were washed 3 times with PBST, once with PBS and finally with ddH2O before centrifugation at 200 x g for 5 min to dry. Slides were scanned at 635 nm using a GenePix® 4300A scanner to detect bound HA-tag antibody. Images were analysed using image analysis software GenePix® Pro 7, which automatically detected spots and identified proteins according to the print layout. The local background signal surrounding each spot was also read to enable background correction for each spot. Each miniarray was analysed separately, with the mean fluorescence at 635 nm after subtraction of background fluorescence (F635 – B635) calculated for each SH2 target from the five replicate spots. For analysis of Affimer binding specificities, the F365 – B635 calculated for each SH2 protein spot against that Affimer clone was averaged over three 50 experimental repeats. The Affimer was considered to be a positive hit if the signal for the intended target was ≥ 50x that of the signal for the buffer-only control spot. Cross-reactions to other targets were deemed significant if the signal totalled ≥ 10% of the intended target signal.
Purified protein ELISA
Purified protein ELISA were performed to test binding of HA-tagged Affimer proteins to their BAP-tagged SH2 target. Wells of Nunc-Immuno™ Maxisorp™ F96 plates were incubated with 50 µl of 5 µg/ml streptavidin (Molecular Probes® Life Technologies) in PBS at 4°C overnight. Plates were blocked with Blocking Buffer overnight at 37°C, washed with PBST, and 50 µl of 10 µg/ml SH2 protein in Blocking Buffer added per well. For streptavidin only controls, 50 µl of Blocking Buffer only was added. SH2s were incubated in the wells for 2 h at room temperature, followed by 1 x wash with PBST and incubation with 50 µl of 10 µg/ml Affimer protein in Blocking Buffer, for 1 h at room temperature. Each Affimer was tested against both SH2- containing and streptavidin-only wells. Wells were washed with PBST and incubated with 50 µl HA-tag antibody (1:20,000 ,Abcam, ab119703) in Blocking Buffer, for 1 h at room temperature. After 1 x wash with PBST, wells were incubated with 50 µl anti-mouse-HRP antibody (1:10,000; Abcam, ab6789) in Blocking Buffer for 1 h at room temperature. Plates were washed x 6 with PBST and HRP was detected using SeramunBlau® fast TMB (Seramun Diagnostica GmbH). Absorbance at 620 nm was read after 3 min and 10 min, before the reaction was stopped with 1 M H2SO4 and the absorbance read again at 450 nm.
Cell culture
U-2 OS, HEK293 and HeLa cell lines (ATCC) were maintained in DMEM supplemented with 10% fetal bovine serum and 100U/mL penicillin-streptomycin at 37oC in 5% CO2. The identity of all cell lines was verified by STR and all cell lines were mycoplasma negative.
Plasmid transfections
Affimer DNA was subcloned from pBSTG into pCMV6-tGFP (Origene) using the Affimer-GFP forward and reverse primers. For reverse transfection with 50ng of Affimer DNA using Lipofectamine 2000 (100nl; Invitrogen; HEK293 and U-2 OS cells) or 100ng of Affimer DNA using X-Treme Gene 9 (300nl; Roche; HeLa cells) in 20 uL Opti-MEM were incubated in 96 well Viewpoint plates (PerkinElmer) for 20 mins. 80 uL of cell suspensions were then added (1x104cells/well for HEK293 and U-2 OS cells, and 5 x 103 cells/well for HeLa cells).
pERK Translocation Assay
pERK nuclear translocation was assessed as previously described [29]. Briefly, cells transiently transfected with GFP-tagged Affimer were starved for 1h in serum-free media and stimulated with 25 ng/ml EGF for 5 mins. Cells were rinsed in DPBS and fixed in 4% paraformaldehyde (PFA) for 15 min. Cells were then permeabilised with ice-cold methanol for 10 min at -20oC and rinsed with PBS before blocking in 1% milk for 10 mins prior to incubation with anti-pERK antibody (1:100; Cell Signalling Technology 4370) in 1% milk for 1 h at room temperature. Cells were washed 3 times in PBS and incubated with Alexa-Flour 568 (1:1000; Molecular Probes, Invitrogen) and Hoechst 33342 (1:1000; Molecular Probes, Invitrogen) in 1% milk for 1 h at room temperature. Cells were washed 3 times in PBS and stored at 4oC until imaging. Plates were imaged using ImageXpress® PICO automated cell imaging system (Molecular Devices) and analysed using MetaXpress® High-Content Image Acquisition and Analysis software (Molecular Devices).
Fluorescence Anisotropy
Fluorescence anisotropy (FA) assays were performed on Grb2-SH2 Affimers. All Affimer and Grb2 SH2 samples were dialysed into 50 mM Tris, 100mM NaCl, pH7.4 prior to use. Assays were set up in 96 well plates and analysed using a Tecan Spark™ 10M microplate reader. 20 µM Affimer solutions were set up in triplicate and sequentially diluted by a factor of 2 across 12 wells. A fluorescein isothiocyanate-labelled phosphopeptide (FYp; FITC-GABA-S-pY-V-N-V-Q) was added to these wells to a final concentration of 20 nM. Grb2 SH2 protein was added to wells to a final concentration of 0.25 µM and the anisotropy measured in each well. Polarisation values for each Affimer concentration were plotted using a logarithmic scale (log10) for the concentration values, and the resultant sigmoidal curve fitted using the logistic function on Origin 9.1 software. From this fit, half maximal inhibitory values (IC50) values were calculated automatically by Origin.
Surface Plasmon Resonance
Full-length Grb2 protein was expressed from a pET28a vector using the same method as SH2 domain expression. The protein also contained an N-terminal His tag and no BAP tag. All proteins used in Surface Plasmon Resonance (SPR) were further purified using S.E.C, which also functioned as a method to separate the Grb2 monomer from the dimer. Only monomeric fractions were used in SPR. Grb2 was diluted to 5 µg/ml in 10 mM Sodium Acetate, pH 5.6 and immobilised onto Amine-coupling chips (sensor chip CM5, GE Healthcare). Affimer concentrations of 6.25 nM – 400 nM in 10 mM Sodium Acetate, pH 5.6 were flowed over the immobilised Grb2 at a flow rate of 80 µl/min for 1–3 min in succession and binding was measured. A 1M NaCl wash was used for chip regeneration between measurements. Binding curves were fitted using BIAevaluation 3.2 software and KD values calculated from these. An activated flow cell containing no Grb2 that had been capped using ethanolamine was used as the blank.
Protein extraction, immunoprecipitation and immunoblotting
Protein extraction, immunoprecipitation and immunoblotting were as previously described [50]. Briefly, cells were washed with ice-cold PBS and lysed in Mammalian Lysis Buffer (50 mM Tris; 150 mM NaCl; 1% (v/v) Nonidet P-40 (Sigma); pH 7.4) supplemented with HALT protease inhibitor cocktail and phosphatase inhibitor 2 (SigmaAldrich), for 30 min on ice, followed by centrifugation at 10,000 xg for 10 min at 4°C. Protein concentrations were measured by BCA assay, as per manufacturer’s instructions (ThermoFisher).
For immunoprecipitation mammalian cells lysates, clarified lysates of His-tagged Affimer proteins produced in BL21 StarTM (DE3) E coli., His-Tag Dynabeads (ThermoFisher) and the Kingfisher Flex (ThermoFisher) were utilised. Dynabeads were incubated with 80 µl clarified lysate in 1x blocking buffer (SigmaAldrich) in wash buffer (100 mM Sodium-phosphate, pH 8.0, 600 mM NaCl, 0.02% Tween-20) for 10 min, and rinsed with wash buffer. Beads were then incubated with 500 µg mammalian cell lysate for 90 mins at room temperature. Following three washes, proteins were eluted by incubation in His elution buffer (300 mM Imidazole, 50 mM Sodium phosphate, pH 8.0, 300 mM NaCl, 0.01% Tween-20) for 10 min. Immunoprecipitants were heated in 4xSDS-PAGE Sample Buffer (8% (w/v) SDS; 0.2 M Tris-HCl (pH 7); 20% glycerol; 1% bromophenol blue; 20% β-mercaptoethanol) and run on a 15% SDS-PAGE gels before transfer to nitrocellulose membrane using the BioRad Transblot Turbo. Membranes were then blocked in 5% milk in TBS-T before overnight incubation at 4°C with rabbit Grb2 (1:5,000, Abcam ab32037), or rabbit anti-6xHisTag-HRP (1:10,000 for 1hr at room temperature, Abcam, ab1187). Membranes were rinsed three times with TBS-T before 1 hr incubation at room temperature with goat-anti-rabbit HRP (Abcam, ab97051) if required, followed by three more TBS-T rinses, and development using Immunoblot Forte Western HRP (Millipore), according to the manufacturer’s instructions. Blots were imaged using an Amersham™ Imager 600 (GE Healthcare, Chicago, IL).
Statistical analysis
Statistical analyses were carried out in GraphPad Prism 8.00 software (GraphPad Software, La Jolla, CA), with robust Z scores calculated in Microsoft Excel (Redmond, WA) as per the formulae in Birmingham et al. (2009) [51]. Statistical assumptions of equal variance for one-way ANOVA were tested with Brown-Forsythe tests. Fluorescent anisotropy data was plotted in Origin 9.1 software (OriginLab Corporation, Northampton, MA) and curves fitted with the logistical function.