Computational protocol: Delineation of B-cell Epitopes of Salmonella enterica serovar Typhi Hemolysin E: Potential antibody therapeutic target

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Protocol publication

[…] 20-mer random phage display library was constructed according to Adey and coworkers with slight modifications. Random peptide oligonucleotide inserts and a reverse primer were synthesized (1st Base, Malaysia) with the sequence 5′-CCGGCCATGGCC(NNK)20GCGGCCGCATAGACTGTT-3′ and 5′-AACAGTCTATGC-3′ respectively. The degenerate oligonucleotides, which consist of 20 repeating NNK codon with N being an equal mixture of A, G, C and T, each K is an equal mixture of G and T. This allows the coding of all available amino acids and one stop codon. Each synthesized oligonucleotide was assembled into double stranded DNA by a filling in reaction using Klenow fragment (NEB). The degenerate sequence was cloned into pLABEL phagemid vector with NcoI and NotI restriction sites and introduced into E. coli XL1-Blue competent cells (Agilent Technologies, CA) through electroporation. Approximately 400 transformations were done to construct the random peptide library, and followed by recovery and storage of the library. [...] The sequence of HlyE from S. Typhi was obtained from UniProt (id: Q8Z727) and was further searched against Basic Local Alignment Search Tool (BLAST) for suitable templates in RCSB Protein Data Bank (PDB). Sequence alignment with template (PDB ID: 1QOY) to model the HlyE monomer was performed by Clustal Omega, . The 3D model for the protein was performed by MODELLER 9v14, with a total of 100 models being randomly generated. The model with the best DOPE was evaluated by Ramachandran plot from PROCHECK. This HlyE 3D model generated will serve as input for structural analysis and molecular docking of the identified epitopes. [...] Prediction of linear antigenic regions of HlyE was carried out using BCPREDS web server (http://ailab.cs.iastate.edu/bcpreds/) using an epitope length of 20-mer with a cut off value exceeding 0.8. Putative antigenic determinants were also analysed by the ElliPro prediction web tool (http://tools.immuneepitope.org/tools/ElliPro). Based on the 3D structure of the HlyE protein antigen, linear epitope residues were obtained with default input setting (distance R set at 6 Å; score S set at 0.5). Prediction of continuous antigenic residues of HlyE protein that could possibly interact with the antibodies of interest was carried out through CBTOPE web server which is solely based on the primary sequence of the antigen (http://www.imtech.res.in/raghava/cbtope/). [...] Sequences from the clones obtained from biopanning was filtered using the tools in the SAROTUP suite to exclude any possible TUPs before proceeding as input for mapping and aligned with the HlyE sequence to identify overlapping sequences. Then, the ‘filtered’ peptides were then mapped to the 3D structure of HlyE monomer from S. Typhi using the Pepitope and EpiSearch web tool to analyse the most probable conformational epitopes present on the surface with the respective algorithm and default setting. The potential linear and conformational epitopes were analysed further based on overlapping residues from ElliPro, BCPREDS, CBTOPE, Pepitope and EpiSearch bioinformatic tools. [...] The verified mAbs that specifically bind to both linear and conformational epitopes were subjected to comparative modelling using MODELLER 9v14, to construct 3D scFv models. Multi-template modelling was performed to model the scFv as the sequence identity between templates and target scFv is below 70%. Templates used for the A11 scFv are PDB ID: 5c6w, 2ykl, 5cgy, 3wlw, 4xak, 4gsd, 4ydk, 4osu, 4jdt, 1yrw, 4k91; C2 scFv with PDB ID: 3fno, 3juy, 4fz8, 4hk0, 4lri, 5c6w, 3gkw, 3gnm, 3ld8, 4lf3. A total of 100 models were randomly generated for each respective model. The model with the best DOPE score was evaluated by Ramachandran plot from PROCHECK. Loop refinement was performed by MODELLER to optimize the model with residues situated at disallowed region in Ramachandran plot. A11 and C2 3D models were used for subsequent docking analysis. […]

Pipeline specifications

Software tools MODELLER, PROCHECK
Application Protein structure analysis
Organisms Salmonella enterica subsp. enterica serovar Typhi, Homo sapiens
Diseases Salmonella Infections, Typhoid Fever