Phage display is a revolutionary in vitro technique used to study protein-ligand interactions. Most commonly based on filamentous phage systems, the technology has forever changed antibody discovery and engineering by allowing the screening of vast libraries in short timeframes. In this article, we discuss what is the phage display technology and the main phage systems used for biopanning campaigns. Check our frequently asked questions (FAQs) page about phage display for a complete overview of all steps of this robust process for antibody generation.
The purpose of the phage display technology
Created in 1985 by George Smith, the phage display technology was devised as an in vitro method to study protein-ligand interactions. Smith achieved this purpose by fusing specific polypeptides to surface proteins of lysogenic filamentous phages and subsequently propagating them in a suitable host (i.e. Escherichia coli). Since then, the technology evolved to allow the display of larger proteins like antibodies.
The success of this technology rests on the fact that the phage phenotype and genotype are linked. This property ensures that a single E. coli clone produces identical phage particles and thus allows the creation of vast libraries containing up to 1010 different clones. Subsequent screening of these libraries can be performed in liquid (i.e. in suspension) or semi-solid phases (i.e. phage in suspension against an immobilized ligand) and it primarily allows the enrichment of polypeptides/proteins/antibodies with high affinities towards specific ligands.
Affinity-driven enrichment of protein repertoires has been successfully used in antibody discovery, antibody engineering (i.e. affinity maturation), and epitope mapping that are particularly useful in areas like therapy, diagnostics, and research.
Phage display systems
E. coli filamentous phage M13 containing a circular single-stranded DNA (ssDNA) genome, is the most common choice for antibody phage display. Antibodies in scFv, Fab, or VHH formats are typically fused to surface proteins like the minor coat protein (pIII) and displayed on M13’s surface. This minor protein consists only of 406 amino acids and occurs in the tip of the phage at 3 to 5 copies per particle. It also plays an essential role in phage infectivity by binding directly to E. coli pilus (F factor).
To compensate for the inevitable loss of functionality that occurs when an antibody is fused to pIII, researchers developed the phagemid/helper phage system. Phagemids are simplified variants of M13 containing:
- M13’s origin of replication
- A pIII gene with adequate cloning sites to incorporate different antibody fragments
- An antibiotic resistance gene for selection of positive clones
In contrast, helper phages correspond to the wild-type version of M13 containing all essential elements for phage replication, assembly, and packaging. Moreover, the helper phage contains a slightly defective origin of replication, ensuring that the wild-type and the antibody-fused version of pIII are both displayed on the surface of M13 after assembly.
Phages T7, T4, and lambda – all E. coli phages – are the most common alternatives to M13 for antibody phage display technologies. These systems, although less mature than phagemid systems based on M13, present some advantages that are worth considering.
In general, these alternative phage systems push the boundaries of conventional phage display techniques by allowing larger proteins to be displayed, increasing the success rates of soluble protein display, and allowing the display of higher protein densities. Additionally, phage T4 is particularly interesting due to the ability to fuse protein fragments to non-essential capsid proteins, minimizing the structural burden imposed by the peptides, proteins, or antibodies to be displayed.
However, a single system cannot be viewed as superior to the other. In fact, many studies show that the experimental outcomes depend mostly on the complexity of the protein to be display and results aren’t always consistent with theoretical predictions.
- Bazan, J. et al. Phage display—A powerful technique for immunotherapy. 1. Introduction and potential of therapeutic applications. Hum Vaccin Immunother. 2012; 8(12): 1817–1828. doi: 10.4161/hv.21703
- Marintcheva, B. Phage Display. Harnessing the Power of Viruses. 2018; 133-160. doi: 10.1016/B978-0-12-810514-6.00005-2