Phage-display reveals interaction of lipocalin allergen Can f 1 with a peptide resembling the antigen binding region of a human γδT-cell receptor

Abstract Although some progress has been achieved in understanding certain aspects of the allergenic mechanism of animal lipocalins, they still remain largely enigmatic. One possibility to unravel this property is to investigate their interaction with components of the immune system. Since these components are highly complex we intended to use a high-throughput technology for this purpose. Therefore, we used phage-display of a random peptide library for panning against the dog allergen Can f 1. By this method we identified a Can f 1 binding peptide corresponding to the antigen-binding site of a putative γδT-cell receptor. Additional biochemical investigations confirmed this interaction.

A codon optimized Can f 1 was synthesized (GenScript, Piscataway, USA), cloned into Qiagen´s pQE70 expression plasmid, produced and purified in M15 E.coli according to the manufacturer´s instruction (QIAexpressionist).

Phage display:
Library: Ph.D.-12 Phage Display Peptide Library Kit E8110S (New England Biolabs GmbH, Frankfurt, Deutschland) Materials, general handling of phages and sequencing of inserts was done as described in the Instruction Manual Ph.D Phage Display Libraries from NEB (NEB, Ipswich, USA) Biopanning for the enrichment of lipocalin-interacting phages: Target rCan f 1 was diluted to 100 μg/mL in coating buffer (100 mM NaHCO 3 pH 8.6).150 μL of this solution was used to coat a single well (per target protein) of a 96-wells microtiter plate (Nunc Maxisorb, Thermo Scientific, Vienna, Austria) overnight.The next morning, the coating solution was discarded and replaced by blocking buffer (5 mg/mL BSA in coating buffer).The microtiter plate was blocked for 60 min at 4 °C.Meanwhile, a 100-fold representation of the original Ph.D.-12 library was prepared by diluting 10 μL (corresponding to 10 11 pfu equalling 100 times the library complexity of 10 9 individual clones) in 90 μL of TBS-T (TBS + 0.1 % Tween 20).Next, the blocking solution was discarded and the microtiter plate rapidly washed 6 times in 200 μL TBS-T buffer.The prepared phage library was added to the plate and incubated on a rotating shaker at room temperature (RT) for 60 min.Then, the phage suspension was discarded and the plate washed again 10 times in 200 μL TBS-T to remove non-binders.Finally, bound phages were eluted competitively in 100 μL solution of the respective free target (100 μg/mL Can f1 in TBS) on a rotating shaker at RT for 60 min.Panning procedure was repeated three times.

Binding studies
3.1.Can f 1-ANS binding studies 1 μM delipidated Can f 1 (20 μg/mL) were mixed with a range of ANS concentrations (0, 1, 2, 5, 10 and 20 μM or -fold molar excess respectively) in 20 mM potassium phosphate buffer pH 7.2 containing 0.86 % acetonitrile at RT. Acetonitrile was later used as a solvent for the synthetic peptides and was therefore already included in this experiment to rule out any adverse effects.Samples (100 μL per well) were prepared in triplicates and transferred to black half area 96-wells microplates (Greiner Bio-One, Rainbach, Austria).After 10 min incubation in the dark, fluorescence measurements were performed on a Fluostar OMEGA plate reader (BMG Labtech, Ortenberg, Germany) with OMEGA and MARS software.Can f 1-ANS complexes were excited at 355 nm and their fluorescence recorded at 460 nm.A second series of measurements omitting Can f 1 was performed under identical conditions to establish ANS background signal.

ANS displacement from
Can f 1 by palmitic acid ANS was displaced from its complex with Can f 1 (both at 1 μM) by increasing concentrations of the hydrophobic ligand palmitic acid.A series of 50-fold palmitic acid stock solutions in EtOH was prepared -1000, 750, 500, 250, 100 and 50 µM as well as an EtOH blank.The reaction mixture contained 4 µL 500 µg/mL delipidated Can f 1, 1 µL 100 µM ANS, 20 mM potassium phosphate pH 7.2 (see above), 2 µL of respective 50-fold palmitic acid stock and ddH2O to 100 µL.Measurements were performed as described above.

Synthesis and cloning of δ TCR CDR3 peptide and cloning into SH2-fusion protein plasmid
Synthesis of the gene encoding the δ TCR CDR3-peptide SWGVPSNFLLIVSDKLIFG was performed by PCR using two codon-optimized overlapping primers, (forward: 5'-ctGGATCCatgggcagctggggtgtgccgtctaactttctgctgatcgtgagcgacaaactgattttc-3'and revers: 5'-cgCTCGAGtcattatttttcaaactgcggatggctccacgcagagccgaaaatcagtttgtcgctca-3'), which was then cloned into pASK75 adding a C-terminal Strep-tag onto the construct.It was afterwards subcloned into a SH2 domain containing pET21d+ plasmid.The resulting gene encoded a mature protein combined of an N-terminal Strep-tag, the SH2-fusion partner, the CDR3-peptide (slightly modified to accommodate an additional EcoRI site for control digestion) and a Cterminal His-tag (Supplementary Figure S2).

Expression, purification of SH2-CDR3 fusion peptide under denaturing conditions and renaturation
A single colony of Rosetta(DE3) cells harbouring pET21d+ SH2-CDR3 plasmid was used to inoculate 12 mL of LB medium (containing 100 μg/mL ampicillin and 25 μg/mL chloramphenicol).The culture was grown overnight at 37 °C on a rotating shaker (x180 rpm).The next morning, 10 mL of overnight culture were used to inoculate 250 mL of fresh LB medium (+ antibiotics).At an OD 600 of 0.5 protein expression was induced by the addition of 1 mM IPTG for 4 h.Bacteria were harvested by centrifugation at 6,000 g for 20 min and resuspended in 25 mL PBS buffer (supplemented with Roche complete protease inhibitors).Bacteria were lysed by three cycles of French Pressing (at 14,000 psi in an HTU, Digi-F-Press, Heinemann).The resulting lysate was clarified by centrifugation at 12,000 g for 20 min.Then, the supernatant was discarded and the insoluble pellet -containing the recombinant proteinwas solubilised in 10 mL 8 M urea in PBS under continuous mixing at room temperature for 2 h.The extract was passed through a 1 mL bed volume Ni-NTA Agarose (Qiagen) gravity flow column to capture the SH2-CDR3 fusion protein via its C-terminal His-tag.To allow on-column refolding, the urea concentration was reduced over the course of several washing steps: The higher urea buffer was slowly replaced by applying 5 mL of the next lower concentration carefully on top of the Ni-NTA agarose bed and allowing complete flow-through by gravity.This procedure was repeated for a total of 5 times, thereby reducing the initial 8 M urea concentration stepwise to 4, 2, 1, 0.5 and 0.25 M. The purified and refolded fusion protein was finally eluted in 3 mL PBS buffer containing 250 mM imidazole.Samples collected during protein expression, solubilisation and purification were analysed by 12 % SDS-PAGE (see Figure S1).
A control experiment omitting Can f 1 coating was performed to determine unspecific binding and background signal.Binding of SH2-CDR3 to Can f 1-coated wells was competed against using 10-fold molar excess of the original Can f 1-binding phage-display peptide KLWSIPTNFLLP.All experiments were performed in triplicates.