Construction of an Ultra-Large Phage Display Library by Kunkel Mutagenesis and Rolling Circle Amplification

• DOI: 10.1007/978-1-0716-3381-6_10
• Publication Date: 2023-09-07T18:02:12Z
• Authors: Renhua R. Huang; Michael Kierny; Veronica Volgina; Makio Iwashima; Christina Miller; Brian K. Kay
• Keywords: SARS-CoV-2; Mutagenesis; Humans; COVID-19; Bacteriophages; Gene Library

An important contributor to the successful generation of recombinant affinity reagents via phage display is a large and diverse library. We describe, herein, the application of Kunkel mutagenesis and rolling circle amplification (RCA) to the construction of a 1.1 × 1011 member library, with only 26 electroporations, and isolation of low- to sub-nanomolar monobodies to a number of protein targets, including human COP9 signalosome subunit 5 (COPS5), HIV-1 Rev. binding protein-like protein (HRBL), X-ray repair cross-complementing 5/6 (Ku70/80) heterodimer, the receptor-binding domain (RBD) of SARS-CoV-2, and transforming growth factor beta 1 (TGF-β1).