The porcine jejunum epithelial cell line IPEC-J2 (DSMZ, Braunschweig, Germany) was maintained in DMEM/Ham's F-12 medium mixture (Gibco Life Technologies, GrandIsland, NE, USA) supplemented with 5% heat-inactivated fetal bovine serum (Invitrogen, GrandIsland, NE, USA), 1% insulin-transferrin-selenium-X (Gibco, Waltham, MA, USA), and antibiotics (penicillin/streptomycin). The HEK 293 LTV cell line (Cell Biolabs, San Diego, CA, USA) was cultured according to the manufacturer's instructions. IPEC-J2 and HEK 293 LTV cells were grown at 37 °C in an atmosphere of 5% CO2.
Construction of porcine TLR2-expressing IPEC-J2 cells
The porcine TLR2 gene was amplified from pUNO1-pTLR02 (InvivoGen, San Diego, CA, USA) by PCR using the designed primers (Supplementary Table S1). The primers included restriction enzyme sites, and additional sequences were inserted to remove the stop codon in the TLR2 sequence and to fit in the eGFP frame for the C-terminal eGFP-tagged TLR2 expression. PCR was initiated with a 4-min denaturation at 95 °C, followed by 5 cycles of 30 s at 95 °C, 30 s at 43 °C, and 2 min at 72 °C; 30 cycles of 30 s at 95 °C, 30 s at 62 °C, and 2 min at 72 °C. The PCR product was inserted into a lentiviral transfer plasmid pWPXL (Addgene, Watertown, MA, USA) by Pme/Mlu sites to generate pWPXL-pTLR2-eGFP.
To produce lentiviruses, three plasmids were used: the pWPXL lentiviral expression plasmid, the PsPAX2 packaging plasmid (Addgene), and the pLP/VSVG envelope plasmid (Invitrogen, Carlsbad, CA, USA). The porcine TLR2-expressing lentivirus was prepared as follows. Three hours before transfection, the cell medium was replaced with 25 µM chloroquine-containing medium. Then, 26.5 μg of pWPXL-pTLR2-eGFP, 9.2 μg of PsPAX2, and 5 μg of pLP/VSVG were transfected into 80% confluent HEK 293 LTV cells using the calcium phosphate precipitation method. After 12 h of transfection, transfected cells were treated with 15% glycerol solution and incubated for 24 h. Culture supernatants were harvested every 12 h and filtered using 0.45 µm pore filters (Sartorius, Republic of Korea). To precipitate lentiviruses, PEG 10000 and NaCl were added to the lentiviral supernatant and concentrated by centrifugation at 15,000 × g for 2 h at 4 °C. The virus pellet was dissolved in DMEM and stored at -70 °C.
For lentiviral transduction, IPEC-J2 cells were seeded at a density of 1 × 105 cells/well and grown to 60% confluence in a 35-mm culture dish before transduction. Cells were treated with lentiviruses in the presence of 8 μg/mL polybrene (Sigma-Aldrich, St. Louise, MO, USA). Forty-eight hours after transduction, the medium was replaced with fresh media, and transgene expression was observed by fluorescence microscopy.
Evaluation of lentiviral transduction
To evaluate eGFP expression in the transduced IPEC-J2 cells, flow cytometric assays and western blotting were performed. For flow cytometry, cells were detached using 0.25% trypsin and fixed with 4% paraformaldehyde. Fixed cells were analyzed using FACS Aria II (BD Biosciences, San Jose, CA, USA).
For western blotting, the transduced IPEC-J2 cells were lysed using RIPA buffer (Sigma-Aldrich) with protease inhibitor and phosphate inhibitor (Roche, Basel, Switzerland). Equivalent amounts of protein samples were electrophoretically separated, and the proteins were transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk and 0.05% Tween-20 in TBS for 1 h at room temperature and incubated overnight at 4 °C with HRP-conjugated rabbit anti-GFP polyclonal antibody (Santa Cruz Biotechnology) (1:500). After washing three times with 0.05% Tween-20 in TBS, the membrane was reacted with ECL detection reagents (Santa Cruz Biotechnology) and analyzed using a luminescent image analyzer LAS-3000 (Fujifilm, Japan). Full-length blot image was presented in Figure 2A.
To evaluate porcine TLR2 expression in transduced IPEC-J2 cells, a ligand binding assay was performed. IPEC-J2 cells were seeded and grown to 60% confluence in 35 mm confocal glass bottom dishes (SPL, Republic of Korea). Cells were treated with pTLR2-eGFP carrying lentiviruses in the presence of 8 μg/ml polybrene (Sigma Aldrich) and incubated for 48 h. Then, 10 μg/mL of Texas Red-labeled Zymosan A from Saccharomyces cerevisiae (Molecular probes, Eugene, OR, USA) was incubated for 1 h at 37 °C and washed three times with PBS. Cells were then fixed in 4% PFA and analyzed using confocal microscopy (TCS SP8X, Leica, Wetzlar, Germany).
Cell-based phage display
The Ph.D.-C7CTM Phage Display Peptide Library Kit (New England Biolabs, Ipswitch, MA, USA) was used for two types of biopanning: biopanning with and without a subtractive round (Fig. 1). In non-subtractive biopanning, after transduction with pTLR2-eGFP-carrying lentiviruses, IPEC-J2 cells were harvested with ice-cold PBS (without Ca2+ and Mg2+) containing 5 mM EDTA. The harvested cells were then washed with DMEM containing 1% BSA, and cell numbers were counted. Then, 1 × 106 cells were prepared in DMEM containing 1% BSA and 2 × 1011 pfu of the phage library (10 μL of the phage library) were added to the cell suspensions. Cells were incubated for 2 h on ice to prevent endocytosis of any bound phage. To remove unspecific phages, the cell pellet was washed three times with 500 μL of PBS containing 1% BSA and 0.05% Tween 20 by gentle pipetting. At the end of the final wash, 1 mL of 0.1 M glycine-HCl (pH 2.0) was added to the cell pellet to elute the cell-bound phage. After vortexing for 3 min, the tube was centrifuged for 3 min at 4°C. The supernatant containing the eluted phage was transferred into a new 1.5 ml tube and neutralized with 60 μL of 2 M Tris base. Eluted phages were quantified by titration using phage-plaque-forming assay after infection into Escherichia coli and also used for DNA sequencing to determine their peptide sequences according to the manufacturer's instructions (New England Biolabs). The above steps were referred to as 'one round of biopanning', and an equal amount of phage was used for the consecutive another two rounds of biopanning after being amplified in E. coli.
The experimental procedures of biopanning with a subtractive round were the same as the above biopanning without a subtractive round except for one process of negative selection. In negative selection, 2 × 1011 pfu of phage was added to 1 × 106 wild-type IPEC-J2 cells for 30 min on ice. After centrifugation, cell pellets containing cell-bound phages were discarded to remove the wild-type IPEC-J2 cell-bound phage. The recovered supernatant was then directly added to 1 × 106 lentiviral-transduced IPEC-J2 cells, and three rounds of positive selection were performed.
Phage sequencing and screening false positives
After the third round of biopaning in each non-subtractive and subtractive cell-based phage display, ssDNA from the eluted M13 phages was isolated using the NaI/EtOH precipitation method 22. Then, 50 ng of isolated phage ssDNA was amplified using primers flanking the variable region at the N-terminus of the pIII gene of the phage. The PCR reaction contained 10 × PCR buffer, 25 mM MgCl2, 2.5 mM dNTP, 1.5 U Ex-Taq polymerase (Takara, Kyoto, Japan), and 10 pmol of forward and reverse primers. Thirty-five cycles (10 s at 98 °C, 20 s at 58 °C, and 30 s at 72 °C) were performed. PCR products were purified from a 2.5% agarose gel using a QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). Forty nanograms of DNA per sample was pooled together in a volume of 100 μL.
A phage ssDNA library for Illumina sequencing was constructed using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA) according to the manufacturer's recommendations. The prepared libraries were then sequenced using an Illumina HiSeq 2000 for 100 bp paired-end reads. Adapter sequences of the reads were trimmed with Cutadapt 1.10 23, and the sequence reads were quality-filtered using in-house Perl scripts 24,25. In brief, when 95% of the nucleotide bases in a read were given a quality score of over 31 (Illumina 1.8+) and the read length was ≥70 bp, the read was used in the next step. The sequence reads that were precisely matched with the two nucleotide sequences consisting of the flanking region of the phage display variable region (upstream: 5’-TATTCTCACTCTGCTTGT-3' and downstream 5’-TGCGGTGGAGGT-3') were retained for further processing. Nucleotide sequences of phage display variable regions for each sample were extracted, and the variable regions encoding each unique peptide sequence were counted for each sample. The relative abundance of each peptide was calculated as the fraction of total reads in the library that encoded the peptides.
False positives were screened using two web-based programs, SAROTUP and PepBank, with default options. SAROTUP was screened for identification of ‘non-target sequences’ which could be frequently selected irrelevant peptide sequences with target binding during phage display, such as plastic binders and sequences having propagation advantages during amplification in E. coli 26. PepBank was searched for sequences already reported from previous researches 27.
Validation of identified peptide ligands
To evaluate the binding ability of the peptides to TLR2, peptide binding assays and ligand competition assays were performed. First, peptides for the assays were synthesized with a purity of > 95% (Peptron, Republic of Korea). The peptides included a consensus motif (CX7C, seven amino acids from each selected peptide with two cysteines back and forth, a total of nine amino acids with a single intra-disulfide bond), rhodamine B conjugated as the fluorescence label, and alanine and glycine residues at N-terminal and C-terminal of the peptide, respectively, to increase the stability of the synthetic peptides.
For the peptide binding assay, IPEC-J2 cells and lentiviral-transduced IPEC-J2 cells were incubated with 2 nM, 20 nM, 200 nM, and 2 μM of each peptide. Cells were then washed three times with PBS for 3 min and fixed in 4% PFA. Fluorescence was measured using a FACS Aria II (BD Biosciences).
For the ligand competition assay, IPEC-J2 cells and transduced IPEC-J2 cells were pre-incubated with 2 μM of each peptide for 30 min at 4 °C, and 0, 1, 2, 5, or 10 μg of zymosan A (Thermo Scientific) was added. Cells were then washed three times with PBS for 3 min and fixed in 4% paraformaldehyde. Fluorescence was measured using a FACS Aria II (BD Biosciences).
Results are expressed as the mean ± SD. Statistical significance was determined using Student's t-test and one-way ANOVA. All statistical significance is denoted by * P < 0.05, ** P < 0.01, and *** P < 0.001.
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.