Efficient Cloning of Inserts for Phage Display by Golden Gate Assembly

Abstract

Phage display enables the discovery of high-affinity binders. In phage display, one commonly uses traditional cloning methods to insert DNA into the coding region of one of the five capsid proteins. Here we describe the use of a new vector with kanamycin resistance and BsaI sites for the utilization of Golden Gate cloning into the N-terminus of mature protein III. We also describe the successful pentavalent display of six different inserts: the AviD-tag, the Z-domain of protein A, the Myc-tag, the ALFA nanobody, the BC2 nanobody, and the Flag-tag.

Keywords: Avidity; BsaI; Kanamycin resistance; Nanobodies; NeutrAvidin; Peptide; Protein A; Protein III; Z domain.