Journal: bioRxiv

Article Title: Charged Scanning Mutagenesis as a High Throughput Approach for Epitope Mapping

doi: 10.1101/2025.07.20.665262

Figure Lengend Snippet: (A) Mutagenesis by overlap PCR using the WT-RBD cloned in pUC57, a bacterial vector, as a template and employing a common vector-specific forward primer and three position-specific primers: two mutagenesis primers and a vector-specific barcoding reverse primer. The barcodes are introduced to each position in the first round (1) of PCR, and both vector-specific primers are flanked by a yeast-vector overlapping region that is absent in the bacterial vector. The second round (2) of PCR employs two common primers binding to the flanking region of the previous vector-specific primers, hence preventing WT amplification. (B) The workflow for epitope mapping starts from (1) pooling the barcoded joined PCR products from the second round of PCR from the previous step in equimolar ratio and transforming the pooled library along with the double-digested yeast vector into yeast. The transformed cells are plated to estimate the library diversity. The resulting pool can be stored and also subjected to yeast surface display. (2) Deep sequencing of the displayed library to confirm the correct attachment of the desired barcodes with their target mutations. (3) Library subjected to binding with monoclonal antibodies or polyclonal sera and sorting of the cell populations into different bins based on the binding or expression MFI. (4) Deep sequencing of the barcode region from the sorted populations. (5) Analysis of the deep sequencing results and mapping of the identified epitopes on the structure.

Article Snippet: The RBD sequence was cloned into the pUC57 plasmid with flanking regions of around 100 bp from the pETcon plasmid (Addgene plasmid # 41522).

Techniques: Mutagenesis, Clone Assay, Plasmid Preparation, Binding Assay, Amplification, Transformation Assay, Sequencing, Bioprocessing, Expressing