一．Principle of Phage Display Technology

Phage display technology is to clone the coding gene or target gene fragment of the polypeptide or protein into the appropriate position of the phage coat protein structural gene, and express the foreign polypeptide or protein by fusion with the coat protein under the condition that the reading frame is correct and does not affect the normal function of other coat proteins. The fusion protein is displayed on the phage surface with the reassembly of the progeny phage. The displayed polypeptide or protein can maintain a relatively independent spatial structure and biological activity, which is conducive to the recognition and binding of the target molecule. After a certain period of incubation, the peptide library and the target protein molecules on the solid phase are washed away. The unbound free phages are then eluted with competitive receptors or acid. The eluted phages infect the host cells and then multiply and amplify, and then the next round of elution is carried out. After 3-5 rounds of "adsorption-elution-amplification", the phages that specifically bind to the target molecule are highly enriched. The obtained phage preparation can be used to further enrich the target phages with the desired binding characteristics.

二．Commonly used Phage Display Systems - Single-Stranded Filamentous Phage Display Systems

(1) PIII Display System

Filamentous phage is a single-stranded DNA virus. PI is the secondary coat protein of the virus, located at the tail end of the virus particle, and is necessary for phage infection of Escherichia coli. Each virus particle has 3-5 copies of PI protein, which can be structurally divided into three functional regions: N1, N2 and CT. These three functional regions are connected by two glycine-rich connecting peptides G1 and G2. Among them, N1 and N2 are related to the phage adsorption of Escherichia coli pili and penetration of cell membranes, while CT constitutes part of the phage coat protein structure and anchors the C-terminal domain of the entire PI protein to one end of the phage. PII has two sites for inserting foreign sequences. When foreign peptides or proteins are fused between the signal peptide (SgII) and N1 of PI protein, the system retains the complete PI protein and the phage is still infectious. However, if the foreign peptide or protein is directly linked to the CT domain of PI protein, the phage loses its infectivity. At this time, the infectivity of the recombinant phage is provided by the complete PI protein expressed by the helper phage. PI protein is easily hydrolyzed by proteolytic enzymes, so when there is superinfection with helper phage, each phage can display less than one fusion protein on average, which is the so-called "monovalent" phage.

(2) PVIII Display System

PVI is the main coat protein of filamentous phage, located on the outside of the phage, with the C-terminus binding to DNA and the N-terminus extending out of the phage. Each virus particle has about 2700 PV copies. Pentapeptides can be fused near the N-terminus of PVM, but longer peptide chains cannot be fused because larger peptides or proteins will cause spatial obstacles, affecting phage assembly and making it lose its infectivity. However, when helper phages are involved, wild-type PVI protein can be provided to reduce the valency, and peptides or even antibody fragments can be fused at this time. In addition, there are reports on the study of filamentous phage PVI display system. The C-terminus of PVI protein is exposed on the surface of phage, which can be used as a fusion site for foreign proteins and can be used to study the function of the C-terminal structural region of foreign proteins.

三．Differences between PIII and PVIII

The filamentous phage display system generally fuses foreign polypeptides to the N-terminus of the envelope protein pIII or pVIII. Each virion contains 5 copies of pIII protein, and these five proteins can fuse short peptides without affecting the infectivity of the phage. For the main envelope protein pVIII, each virion contains about 2700 copies, of which about 10% can effectively fuse foreign polypeptides or proteins. Peptides expressed as pIII fusions are low-priced (1 to 5 copies per virion), while polypeptides expressed as pVIII fusions are high-priced (~200 copies per virion). The increased affinity/resistance of this high-price pVIII display is conducive to the screening of ligands with very low affinity, while low-price pIII display limits the screening to ligands with higher affinity.

四．Advantages of M13 Phage

M13 phage and its closely related filamentous phages fd and f1 are non-lytic phages, and they do not lyse the host bacteria during proliferation. This greatly simplifies the intermediate phage purification steps between each round of panning, and a simple PEG precipitation method is sufficient to separate the phage from all other contaminating cellular proteins. Other phages used for phage display, such as T7, T4 and Lambda phage, are lytic phages, and time is needed for purification between each round of panning to avoid contamination of cellular proteins by the panning-amplified phage.

The phage display technology service platform developed by Tek Biotech covers common library types such as natural antibody library, immune antibody library, scFv antibody library, peptide library, etc. At the same time, we also provide bispecific antibody customization and antibody humanization services based on phage antibody library technology, providing you with high-quality antibody library and accurate and efficient screening results.