Classification of Phage Display Systems

introduction

The most common bacteriophages used in phage display are E.coli filamentous bacteriophages (f1, fd, M13) , though T4, T7, and λ phage have also been used. This technique has a lot of applications

1 E.coli filamentous bacteriophages (f1, fd, M13)

A filamentous bacteriophage is a type of phage, defined by its filament-like or rod-like shape. Filamentous phages usually contain a genome of single-stranded DNA and infect Gram-negative bacteria. The family of Ff, M13, fd, and f1 are vital phages which have utility in phage display among which M13 phage is the most generally used.

M13 bacteriophage has a cylindrical shape with a length of 880nm and a diameter of 6nm. It encapsulates a single-strand genome that encodes five different capsid proteins which comprise two groups, major coat proteins (pVIII) and minor coat proteins (pVII, pIX, pVI and pIII).

E. coli filamentous bacteriophages are commonly used for phage display. Most antibodies and peptides are displayed at phage proteins pIII and pVIII, which constructed pIII and pVIII display system. Moreover, hybrid phage system enables displaying large proteins with all five M13 coat proteins as N-terminal fusions with pIII, pVIII, pVII and pIX, and also as C-terminal fusions with pVI, pIII, and pVIII.

M13 bacteriophage

pIII is the protein that determines the infectivity of the virion. It consists of 406 amino acid residues and occurs at the phage tip in 3 to 5 copies. An advantage of using pIII rather than pVIII is that pIII allows for monovalent display when using a phagemid combined with a helper phage. Moreover, pIII allows for the insertion of larger protein sequences (>100 amino acids) and is more tolerant to it than pVIII.

pVIII is the main coat protein of Ff phages, which is expressed by gene 8 and occurs in 2700 copies. Therefore it is used to enhance detection signal when phage displayed antibody associates with antigen. Peptides are usually fused to the N-terminus of Pviii which are usually 6-8 amino acids long . This makes the use of this protein unfavorable for the discovery of high affinity binding partners. Moreover modifications of pVIII are made to increase the efficiency of display onto pVIII, and now there has been great progress.

pVI has been widely used for the display of cDNA libraries, which is an attractive alternative to the yeast-2-hybrid method for the discovery of interacting proteins and peptides due to its high throughput capability. pVI has been used preferentially to pVIII and pIII for the expression of cDNA libraries because one can add the protein of interest in the C-terminus of pVI without greatly affecting pVI's role in phage assembly.

pVII and pIX, located to the phage tip opposite that of pIII, may both complement current phage display systems and be used as alternative scaffolds for display and selection to further improve phage display as the ultimate combinatorial engineering platform.

2 T4 Phage

T4 phage (Enterobacteria phage T4) is a bacteriophage that infects E.coli bacteria. It is a member of the T-even phages, a group including enterobacteriophages T2 and T6. T4 phage is a relatively large phage, at approximately 90 nm wide and 200 nm long. It’s double-stranded DNA genome is about 169 kbps long and encodes 289 proteins. T4 phage is built with three essential proteins: gp23, which forms the hexagonal capsid lattice; gp24, which forms pentamers at eleven of the twelve vertices; and gp20, which forms the unique dodecameric portal vertex through which DNA enters during packaging and exits during infection.

T4 Phage

In addition to the essential capsid proteins, gp23, gp24, and gp20, the T4 capsid is decorated with two nonessential outer capsid proteins: HOC (highly antigenic outer capsid protein) and SOC (small outer capsid protein). Both HOC and SOC are dispensable, and bind to the capsid after the completion of capsid assembly. Null (amber or deletion) mutations in either or both the genes do not affect phage production, viability, or infectivity.

HOC and SOC are dispensable T4 capsid proteins that can be used for phage display of multiple copies of peptides and proteins. The phage T4 HOC, SOC bipartite display system is attractive for the expression of cDNA and display of peptides or proteins at high copy numbers on the phage capsid surface. It could be applied to cDNA expression, displays larger proteins in high copy number and inserts into stop codon on the C-terminal of SOC protein that occurs in 810 copies or N-terminal of HOC protein that occurs in 155 copies[13].Therefore, the phage T4 dual site display emerges as a powerful method with an enhanced immune response in animals for research and development of immunological products.

3 T7 Phage

T7 phage is an icosahedral virus of the Podoviridae family and has a linear double stranded (ds) DNA genome. Similar to T4, bacteriophage T7 possesses a head and tail structure. The icosahedral head, where T7 conserves its dsDNA genome, is composed of 415 copies of capsid gp10, arranged as 60 hexamers on the surface and 11 pentamers at the vertices. There exist two isoforms of major coat protein gp10, gp10A and gp10B, in a nine-to-one ratio, resulted from natural translational frame shift at amino acid 341.

T7 Phage

The minor protein gp10B results from a frame shift in the end of the gene that makes the capsid protein 52 residues longer. Fusion proteins are displayed on protein gp10B C-terminally of the 52 extra residues. So that it can avoid problems associated with steric hindrance. T7 phage particles exhibit high stability under various extreme conditions, including high temperature and low pH, which facilitates effective high throughput affinity elutriation. Recent applications of the T7 phage display system have been instrumental in uncovering mechanisms of molecular interaction, particularly in the fields of antigen discovery, vaccine development, protein interaction, and cancer diagnosis and treatment.

4 lambda phage

lambda phage (Enterobacteria phage λ, coliphage λ) is a bacterial virus which infects the bacterial species E. coli. It was discovered by Esther Lederberg in 1950. The lambda phage has an icosahedral head. The main structure of the shell is built from the major coat protein gpE (415 copies) and is stabilized by the capsid protein gpD (402–420 copies). The head is linked to a flexible helical tail constructed by disks of the major tail protein gpV.

Both the tail protein gpV and the head protein gpD have been used for phage display. Initially, the lambda foo vector was constructed for the C-terminal display on gpV, with a low display level that made it suitable for capturing high-affinity interactions. Later, systems were developed for the display of peptides N-terminally or C-terminally to the major coat protein gpD. The lambda phage has been engineered to display efficiently multiple copies of peptides or even large protein domains providing a powerful tool for screening libraries of peptides, proteins and cDNA.

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