Construction of a T7 phage random peptide library by combining seamless cloning with in vitro translation

Abstract T7 phage libraries displaying random peptides are powerful tools for screening peptide sequences that bind to various target molecules. The T7 phage system has the advantage of less biased peptide distribution compared to the M13 phage system. However, the construction of T7 phage DNA is challenging due to its long 36 kb linear DNA. Furthermore, the diversity of the libraries depends strongly on the efficiency of commercially available packaging extracts. To address these issues, we examined the combination of seamless cloning with cell-free translation systems. Seamless cloning technologies have been widely used to construct short circular plasmid DNA, and several recent studies showed that cell-free translation can achieve more diverse phage packaging. In this study, we combined these techniques to construct four libraries (CX7C, CX9C, CX11C and CX13C) with different random regions lengths. The libraries thus obtained all showed diversity > 109 plaque forming units (pfu). Evaluating our libraries with an anti-FLAG monoclonal antibody yielded the correct epitope sequence. The results indicate that our libraries are useful for screening peptide epitopes against antibodies. These findings suggest that our system can efficiently construct T7 phage libraries with greater diversity than previous systems.