Abstract
In this paper, we describe multivalent display of peptide and protein sequences typically censored from traditional N-terminal display on protein pIII of filamentous bacteriophage M13. Using site-directed mutagenesis of commercially available M13KE phage cloning vector, we introduced sites that permit efficient cloning using restriction enzymes between domains N1 and N2 of the pIII protein. As infectivity of phage is directly linked to the integrity of the connection between N1 and N2 domains, intra-domain phage display (ID-PhD) allows for simple quality control of the display and the natural variations in the displayed sequences. Additionally, direct linkage to phage propagation allows efficient monitoring of sequence cleavage, providing a convenient system for selection and evolution of protease-susceptible or protease-resistant sequences. As an example of the benefits of such an ID-PhD system, we displayed a negatively charged FLAG sequence, which is known to be post-translationally excised from pIII when displayed on the N-terminus, as well as positively charged sequences which suppress production of phage when displayed on the N-terminus. ID-PhD of FLAG exhibited sub-nanomolar apparent Kd suggesting multivalent nature of the display. A TEV-protease recognition sequence (TEVrs) co-expressed in tandem with FLAG, allowed us to demonstrate that 99.9997% of the phage displayed the FLAG-TEVrs tandem and can be recognized and cleaved by TEV-protease. The residual 0.0003% consisted of phage clones that have excised the insert from their genome. ID-PhD is also amenable to display of protein mini-domains, such as the 33-residue minimized Z-domain of protein A. We show that it is thus possible to use ID-PhD for multivalent display and selection of mini-domain proteins (Affibodies, scFv, etc.).
Highlights
Phage display (PhD) is a powerful method for selection of peptide ligands from a diverse, random population (McCafferty et al, 1990, 1991; Scott and Smith, 1990)
We have previously shown that the censorship in phage libraries can arise even due to small differences in growth rates
ID1-PhD and ID2-PhD Cloning Vectors Figure 1 summarizes the structure of the pIII protein of M13 phage as C-terminal (CT) and two N-terminal (N1 and N2) domains separated by two linkers
Summary
Phage display (PhD) is a powerful method for selection of peptide ligands from a diverse, random population (McCafferty et al, 1990, 1991; Scott and Smith, 1990). Noren and co-workers (Brammer et al, 2008; Nguyen et al, 2014) identified a large number of sequences with random mutations in un-translated regions (UTR) of gene pII; these mutations equip clones with increased growth rate. Enrichment of these fast growing “parasite” sequences is one of the mechanisms for sequence-independent censorship/loss of diversity. This observation suggested that the frequency of spontaneous deletions of a 40 amino acid sequence (1 in 100) is more frequent than deletion of a 14 amino acid sequence (1 in 106, Figure 2) This spontaneous deletion is likely to be the limiting factor of ID-PhD technology for display of large proteins. TEV digestion can be performed on any sequence and is conceptually simple because it requires only measurement of titer before and after addition of TEV protease
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