In the field of life sciences, there are two extremely important biological macromolecules that have been studied by scientists, one is a protein that performs most of the life activities, and the other is a nucleic acid that carries genetic information. Before the advent of phage display technology, the ability to decode nucleic acid sequences was far better than the ability to decode protein sequences, whether it was cost, throughput, or efficiency. After scientists use phage display technology to convert protein-related information into nucleic acid information, they can interpret protein information through high-throughput sequencing, which also provides a more convenient method for protein research.
The principle of phage display technology is to associate the sequence of protein/antibody or small peptide displayed on its surface with the nucleic acid sequence encoding them, and use the super amplification ability of the phage to build a small peptide or antibody library, and the diversity can reach more than 109; Then, phage display technology is used to obtain affinity peptides or antibodies for specific objects through multiple rounds of scouring.
So how was phage display technology discovered and developed? This starts with bacteriophages.
Bacteriophages are a class of viruses that, as the name suggests, are viruses that devour microorganisms such as bacteria, fungi, and superbugs. In nature, bacteriophages are everywhere, and their extreme diversity continues to subvert the "rules of the game" in the field of life, and are developed by biological scientists as the "dark matter" of the living universe and the "nuclear weapons" for the prevention and control of pathogenic microorganisms. In addition to this, phage display is an important area of its application.
In 1985, Professor G. P. Smith inserted a fragment of the EcoR I gene into the BamH I site of the filamentous bacteriophage f1 to transform E. coli, by fusing with the structural protein of the f1 phage, successfully displayed EcoR I ("face") on the surface of the phage (1). Since then, with the construction of a large number of phage display libraries, the establishment of genetic manipulation systems, and the expansion of display platforms and systems, phage display technology has played an important role in different fields, especially protein and antibody related fields.
There are various classification criteria for phage display techniques. According to the different phages, it can be divided into M13, T7, T4, lambda and other systems; According to the different vectors, it can be divided into bacteriophage vector (True Phage) and bacteriophage vector (Phagemid); According to different libraries, it can be divided into random peptide library, cDNA library, antibody library, protein library, etc.
M13 phage display system
Single-stranded filamentous bacteriophages (M13) are filamentous bacteriophages containing single-stranded DNA genomes with tubular capsid proteins, usually consisting of several thousand copies of the primary capsid protein (PVIII.) and the secondary capsid protein (PIII) located at the tail end. Due to the small molecular weight of this protein, it is only suitable for displaying exogenous short peptides. Exogenous peptides that are too large will affect viral packaging and cannot form functional bacteriophages. This system is well suited for screening ligands with low affinity.
T7 phage display system
The T7 phage genome is linear double-stranded DNA, and its capsid protein is usually in two forms, 10A (344 amino acid residues) and 10B (397 amino acid residues), and the 10B capsid protein region is present on the phage surface, so it is used to build a phage display system. Compared with the M13 system, T7 bacteriophages directly lyse the host bacteria without going through the secretion process, and are widely used to screen proteins with different molecular weights and different affinities.
T4 phage display system
T4 phage genome DNA is double-stranded, arranged in a loop, phage capsid has two non-essential coat proteins SOC (9 ku) and HOC (highly antigenic outer capsid protein, 40 ku), so the protein it expresses does not require complex protein purification, can display various sizes of peptides or proteins, rarely restricted.
λbacteriphage display system
The lambda phage is a mild bacteriophage of the long-tailed phage family with an icosahedral head 55 nm in diameter and an elongated tail filament at the end. A linear double-stranded DNA molecule with a genome of 48.5 kb with sticky ends, i.e., 12 nucleotides extending a single strand, and the linear genome can be cycled immediately after infection. The head of the bacteriophage is composed of D protein and V protein, which can build a display system for D protein and V protein. λbacteriophages are assembled in the host cell, without secreting foreign peptides or proteins outside the bacterial cell membrane, and can display active macromolecular proteins (more than 100 kDa) and host cell toxic proteins, and have a wide range of applications.