Efficient Cloning of Inserts for Phage Display by Golden Gate Assembly.

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.

Expanding the chemical diversity of M13 bacteriophage.

Bacteriophage M13 virions are very stable nanoparticles that can be modified by chemical and genetic methods. The capsid proteins can be functionalized in a variety of chemical reactions without loss of particle integrity. In addition, Genetic Code Expansion (GCE) permits the introduction of non-canonical amino acids (ncAAs) into displayed peptides and proteins. The incorporation of ncAAs into phage libraries has led to the discovery of high-affinity binders with low nanomolar dissociation constant (K D) values that can potentially serve as inhibitors. This article reviews how bioconjugation and the incorporation of ncAAs during translation have expanded the chemistry of peptides and proteins displayed by M13 virions for a variety of purposes.