An Overview of Phage Display Systems

1. An Overview of Phage Display Systems By Creative Biolabs

2. Introduction Phage display technology is essentially a technology for screening antibodies. Compared with the production of monoclonal antibodies with immunized animals, phage technology is rapid, simple and efficient, and can be used for the production of humanized antibodies, which have higher application value. Thus, it seems of significant importance to develop phage display systems. There are three common display systems in basic biological research.

3. 1. M13 Filamentous Phage Display System In the single chain M13 filamentous phage display system, filamentophages are a large family of viruses capable of infecting negative bacteria. They contain single chain DNA genomes and tubular capsid proteins, and usually consist of thousands of copies of main capsid protein (PⅧ) and the minor capsid proteins (PⅢ) located at the terminus. This M13 bacteriophage system is divided into four systems: PⅢ, PⅧ, PV and D protein.

4. 1.1 PⅢ PⅢ is the minor capsid protein of filamentous phages, located at the terminus of the virus particles. It is necessary for E. coli phage infection. Each of the virus particles has three to five copies of PⅢ proteins, whose structure can be classified into the three functional domains of N1, N2, and CT3. These domains are linked by G1 and G2, two sections of linker peptides rich in glycine. N1 acts on protein TolA on the membrane of E. coli, related to phage invasion. N2 serves as the receptor binding region, responsible for binding to the F pilus. CT adheres to the bacterial intima before assembly, associated with the suspension of phage assembly.

5. 1.2 PⅧ PⅧ, the main coat protein of filamentous phages, lies in the lateral side of phages. Its C-terminus binds to DNA, with its N-terminus reaching out of phages. Each of the virus particles contains approximately 2,700 copies of PⅧ, in the vicinity of whose N-terminus pentapeptide can be fused. However, it cannot fuse longer peptides, because larger peptides or proteins may give rise to space obstacles, thereby affecting the phage assembly by making it lose infectivity. When helper phages are involved, the wild type of PⅧ protein can fuse polypeptide or even the antibody fragments.

6. 1.3 PV The λ bacteriophage's PV protein makes up the tubular portion of its tail. The tubular structure comes with 32 discoidal substructures, each of which comprises six PV subunits. PV has two folding regions. Exogenous sequences can be inserted into the folding structural domain (the non-functional region) at the C-terminus. The assembly of λ bacteriophages occurs inside cells, which enables the presentation of hard-to-secrete peptides or proteins. The phage display system showed an average copy number of foreign proteins of one molecule per phage, suggesting that foreign proteins or peptides might interfere with the tail-assembly of λ bacteriophages.

7. 1.4 D Protein The D protein engages in the head assembly of the wild-type λ phages. In the case where the mutant phage genome is smaller than 82% of the wild-type genomes, the assembly can be done without D protein. In addition, the assembly of virus particles can be done both in vivo and in vitro. One of the highlights of the system is that the proportion of fusion proteins to D proteins in phages can be regulated by the inhibitor tRNA activity of the host, which is particularly useful for displaying proteins that can cause damage to phage assembly.

8. 2. T4 Phage Display System The T4 phage display system is highlighted by its ability to fuse the two exogenous peptides or proteins in completely different nature with coat proteins, SOC (9 ku) and HOC (40 ku), and directly display them on the surface of T4 phages, avoiding protein denaturation or loss caused by purification. T4 phages are assembled in host cells and do not require a secretory pathway, so they can display peptides or proteins of various sizes. When using recombinant T4 as a vaccine, one can display the target antigen on the surface of an empty capsid, indicating the system’s promising prospect in biosafety.

9. 3. T7 Phage Display System Compared with the M13 system, T7 phage can directly cleave host bacteria, in no need of the secretion process. In the T7 phage display system, phages can be developed within only three hours, saving plenty of time. Besides, for polypeptides with over 50 amino acids, T7 phages can be assembled without helper phages. Importantly, T7 phages perform very stably under the conditions of high PH value, high salt, denatured agents (100 mmol/L DTT), etc. They can be screened under various conditions according to different needs, and maintain high screening activity even after screening.

10. Conclusions Undoubtedly, bacteriophage display systems have many advantages. They achieve the unification of protein/peptide genotype and phenotype, and establish a direct physical link between proteins and their genetic genes. Each phage display system has its own unique advantages, applied for distinct purposes. With the advance of biological science and technology, the existing systems will be optimized and new systems may emerge to fulfill the diverse research and application demands.

11. About Creative Biolabs Creative Biolabs, located in New York, is a leading custom service provider, boasting abundant experience in antibody production and engineering. Its services include mouse and rat, human, monkey, rabbit, chicken, dog, llama and camel monoclonal antibody production. The company also conducts thorough antibody humanization and affinity maturation. In addition, OEM services for large-scale antibody manufacturing are available, too, at the most competitive price in the industry.

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