A Simple Whole-Plasmid PCR Method to Construct High-Diversity Synthetic Phage Display Libraries

Tsoumpeli, Maria T.; Gray, Alison; Parsons, Aimee L.; Spiliotopoulos, Anastasios; Owen, Jonathan P.; Bishop, Keith; Maddison, Ben C.; Gough, Kevin C.

Abstract

Phage display technology utilises peptide and antibody libraries with very high diversities to select ligands with specific binding properties. The production of such libraries can be labour intensive and technically challenging and whilst there are commercial sources of libraries, the exploitation of the resulting binders is constrained by ownership of the libraries. Here, a peptide library of ~ 1 × 109 variants for display on gene VIII was produced alongside three VHH antibody libraries with similar diversity, where 12mer, 16mer or 21mer CDR3s were introduced into the highly stable cAbBCII10 scaffold displayed on gene III. The cloning strategy used a simple whole-plasmid PCR method and type IIS restriction enzyme assembly that facilitate the seamless insertion of diversity into any suitable phage coat protein or antibody scaffold. This method reproducibly produced 1 × 109 variants from just 10 transformations and the four libraries had relatively low bias with 82 to 86% of all sequences present as single copies. The functionality of both peptide and antibody libraries were demonstrated by selection of ligands with specific binding properties by biopanning. The peptide library was used to epitope map a monoclonal antibody. The VHH libraries were pooled and used to select an antibody to recombinant human collagen type 1.

Citation

Tsoumpeli, M. T., Gray, A., Parsons, A. L., Spiliotopoulos, A., Owen, J. P., Bishop, K., …Gough, K. C. (2022). A Simple Whole-Plasmid PCR Method to Construct High-Diversity Synthetic Phage Display Libraries. Molecular Biotechnology, 64, 791-803. https://doi.org/10.1007/s12033-021-00442-4

• Journal Article Type: Article
• Acceptance Date: Dec 17, 2021
• Online Publication Date: Feb 2, 2022
• Publication Date: 2022-07
• Deposit Date: Mar 10, 2022
• Publicly Available Date: Mar 10, 2022
• Journal: Molecular Biotechnology
• Print ISSN: 1073-6085
• Electronic ISSN: 1559-0305
• Publisher: Springer Verlag
• Peer Reviewed: Peer Reviewed
• Volume: 64
• Pages: 791-803
• DOI: https://doi.org/10.1007/s12033-021-00442-4
• Keywords: Molecular Biology; Applied Microbiology and Biotechnology; Biochemistry; Bioengineering; Biotechnology
• Public URL: https://nottingham-repository.worktribe.com/output/7395510
• Publisher URL: https://link.springer.com/article/10.1007/s12033-021-00442-4