T4 Phage as a Phage Display Platform

T4 phage and related strains were used in the initial formulation of many basic biological concepts. These include the clear identification of nucleic acid as a genetic material through the fine structure mutation, recombination, and gene function analysis definition, proving that the genetic code is triple, the discovery of mRNA, restructuring and the importance of DNA replication, dependence and light dependent DNA repair mechanisms, constraints and modification of DNA in prokaryotes from intron/exon arrays from splicing, translation bypass, etc. In view of the various functions of T4 phage, Creative Biolabs provides a comprehensive service for T4 phage display library construction.

Fig 1. T4 phage.

T4 Phage Capsid and Nucleic Acid

T4 phage belongs to the Myoviridae family of the order Caudovirales, which is a large, tailed, double-stranded DNA (dsDNA) virus. T4 phage has an exclusively lytic lifecycle. Mature T4 virion particles containing 172 kbp genome DNA and about 50 kinds of proteins are composed of prolate head and tail, the tail has a shrinkage sheath, the end is a substrate, and the substrate adhesion 6 long tail fibers.

T4 phage capsid protein surface package contains two kinds of adornments—HOC (high antigenic outer capsid protein: molecular weight, 40 kDa) and SOC (small outer capsid protein: molecular weight, 9 kDa), located in an icosahedral symmetry position (160 copies of HOC and 960 copies of SOC per capsid particle). Both proteins are nonessential, that is, not essential for phage infection and activity. They are incorporated into the capsid surface after the completion of capsid assembly.

T4 long tail fiber is composed of four different proteins. Their gene products are gp34, gp35, gp36, and gp37. These proteins form proximal and distal half-fiber segments of approximately 70 nm that hinge at an angle of approximately 160°. Proximal half-fiber consists of gp34 trimer, followed by the formation of hinged or "kneecap" gp35 monomer, and distal half fiber contains gp36 trimer and gp37 at the far end of the trimer. Gp34 and gp37, as well as the short-tail fibrin gp12, require the molecular chaperone gp57 for proper trimeric assembly. gp38 is also required for gp37. Host recognition through long tail fiber tips and lipopolysaccharide hole or outer membrane protein reversible interactions occur. In the region of the phage collar, fibrin (gp wac) is positioned for the phage collar whiskers. They formed six fibers radiating from the neck of the phage. In the process of phage morphogenesis, the whisker with long tail fibers promotes its adhesion to the phage base plate.

Phage Display on the T4 Particles

Phage display is a research field with exponential growth. It has had a major impact on immunology, cell biology, drug discovery, and pharmacology. In addition, plant science is becoming increasingly important. In this technique, the phage capsid is modified with a foreign peptide or protein. It is useful to insert the coding protein specific DNA fragment encoding the phage capsid protein gene. Then, during assembly, fusion proteins are produced and incorporated into phage particles. Phage display technology is a very effective tool to select peptides or proteins with specific binding properties from a large number of variants. Briefly, phage display refers to the presentation of a large number of exogenous (non-phage) peptides, proteins, or antibody fragments on the surface of phage particles.

SOC and HOC protein acts as the anchor protein, both in the T4 phage display system. Recently, it has been reported to show a macromolecular complex on T4—two bipartite fusion proteins were constructed—anthrax lethal factor (LF) fused to HOC, and the N-terminal domain of LF (LFn) coupled to SOC. Some researchers reported a macromolecular complex display system first described using phage T4, which is modified by two controllable outer capsid proteins, HOC and SOC. Sequential assembly of HOC and SOC was performed by first saturating the binding sites of HOC and SOC by attaching LF-HOC and/or LFn-SOC to HOC-SOC-phage using defined in vitro binding systems. Through the LFn with capsid exposure (lethal) structure domain specific interaction, trypsin gap PA63 h (the heptamer protective antigen) is assembled into the heptamer. The assembly of the triple anthrax toxin was then completed by linking EF, the edema factor, to the unoccupied site of the PA63 heptamer. Electron microscopy showed that each capsid was decorated with a layer of heptamer PA63 rings. Up to 229 anthrax toxin complexes, equivalent to 2,400 protein molecules and approximately 133 MDa, 2.7 times the mass of the capsid, were immobilized on a single particle, making it the highest density reported for the virus. This work shows that the T4 capsid lattice provides a stable platform, allowing maximum variations in in vitro source oligomeric complex and for the development of new vaccines, analysis of protein and protein interactions, and a profound understanding of the complex structure of the test.

Reference

1. Gamkrelidze, M. Dąbrowska, K., T4 bacteriophage as a phage display platform. Arch Microbiol, 2014. 196(7): p. 473-479.

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