pSEX81 phagemid Vector

Phage display is a powerful technique for drug discovery in biomedical research, especially antibody libraries. However, there are some technical challenges to the selection process. For example, during the panning step, it is critical to successfully elute antigen-bound phage to avoid losing the most promising binders. Here, we present an efficient protocol for building, screening, and selecting synthetic libraries of domain antibodies using phage display.

The vNAR antibody scaffold used to synthesize the shark antibody library was based on HEL-5A7, a type I vNAR generated by Helen Dooley from an immune library directed against lysozyme. This framework was chosen for its high stability and functional expression in prokaryotic systems. Its 5′ and 3′ ends were flanked by NcoI and NotI restriction sites, respectively, for subsequent cloning into the pSEX81 phagemid vector.

The amplified vNAR fragment and the pSEX81 vector were digested with NcoI and NotI and ligated to construct a recombinant phagemid (Figure 1A①). The ligated phagemids were then transformed into electrocompetent E. coli TG1 and plated on 2X TY-GA agar plates (Figure 1A②–③). The collected clones were used to infect with helper phage and generate a phage antibody library (Figure 1B④). After infection, the phage present in the amplified supernatant are precipitated and ready for selection of binding agents (Figure 1B⑤). After three to four rounds of panning, 10⁹ phages from each round were used in a polyclonal phage ELISA to assess enrichment (Figure 1C⑥). Clones were isolated from the enriched panning wheel, grown overnight, and produced as soluble fragments in a 96-well plate format (Figure 1C⑦). Identification of antigen-specific monoclonal binders by ELISA (Figure 1C⑧). Finally, positive clones were sequenced and analyzed. Based on the ELISA results, suitable clones can be selected and subcloned into prokaryotic or eukaryotic expression plasmids for protein production.

Figure 1. Schematic representation of the improved protocol. (Solemani Zadeh A, et al., 2019)