Alpha Lifetech Inc. has extensive experience in library construction, and it's professional in generating filamentous M13 phage display libraries, including polypeptide libraries, antibody libraries, and others.
Filamentous Phage Display Library
In comparison to previous techniques, the display technology based on filamentous phages like lambda-phage screening has the following advantages: (a) as many as 1010–1011 individual clones can be screened in a single experiment, which could increase the chance of isolating (auto)antigens; (b) the screening of the cDNA library is carried out in a fluid phase, denaturation of the proteins, which may occur on solid surfaces, is minimized; (c) low amounts of sera are required to carry out all the selection procedures; (d) the isolated phages carry the genes encoding the binders of interest, and it is simple to locate and alter these genes in order to conduct additional studies.
M13 Phage Display Library
M13 is one of the Ff phages (fd and f1 are others), a family filamentous bacteriophage (inovirus) member. Ff phages are made of circular single-stranded DNA (ssDNA), which in the case of the M13 phage is 6407 nucleotides long. The genome of M13 is encapsulated in approximately 2700 copies of the major coat protein p8, and capped with about 5 copies each of four different minor coat proteins (p3 and p6 at one end and p7 and p9 at the other end). The minor coat protein p3 binds to the receptor at the tip of the F pilus of the host Escherichia coli. The life cycle is relatively short, with the early phage progeny exiting the cell ten minutes after infection. Ff phages are chronic phages that release their progeny without killing the host cells. In comparison to regular lysis plaques, the infection causes turbid plaques of intermediate opacity on E. coli lawns. However, a decrease in the rate of cell growth is seen in the infected cells. M13 plasmids are used for many recombinant DNA processes, and the virus has also been employed for phage display, directed evolution, nanostructures, and nanotechnology applications.
M13 phage is 895-900 nm long, 6 nm thick, and has a 2.5 nm-wide central core.
The 6407 nucleotides long DNA is packed in linear helical form, and at one end, it contains a 100 nucleotides long hairpin-like structure; it is called intergenic region (IG). This region contains signals for viral packaging and also contains origin sequences for minus strand as well as plus strand synthesis.
All along the 6407 nucleotides, genomic DNA is enclosed by protein subunits called gp8, which are organized in a shingle fashion. The gp8 protein subunits have basic and acidic amino acids ends with their charges and hydrophobic region in the middle. The basic region of the protein is bound to phosphate groups of the DNA backbone. The central hydrophobic core is responsible for protein–protein association. The acidic group is exposed outside. There are 2700 to 3000 subunits per phage particle.
Filamentous phage M13 is widely used to present the desired molecules through coupling to the minor coat protein pIII, pVI, or pVIII of the capsid. For the display of proteins and peptides, N-terminal fusion to the minor coat protein pIII and the major coat protein pVIII proved the most effective, while C-terminal fusion to pVI has been used to display proteins and peptides.
Fig 1 M13 Phage
Fig 2 M13 phage vector
Without changing the sequence, M13 DNA or DNA from any other similar filamentous phages may be utilized to clone foreign DNA into their IG region. There is a limit to how much the phage DNA can grow until the particle size likewise grows.
In order to promote an efficient presentation of fusion proteins in phage particles, we not only use the direct method that inserts the gene of interest into the phage genome but also uses the indirect method of phagemid vector that combines the selected genomic features of the phage with those of bacterial plasmids to generate a fusion to a coat protein-encoding gene (open reading frame of pIII, pVI and pVIII gene). In most cases, using phagemid as a delivery system could be a good choice because phagemids can be modified and contain both phage and bacterial origins of replication, a phage packaging signal, a selectable marker gene, and the gene of the chosen coat protein for fusion. Its use allows easy preparation and maintenance of the vector, high yield of dsDNA, and better transformation rates.
Elements and characteristics of phagemid vector:
1. Coat protein
Filamentous phages have five coat proteins of pIII, pVI, pVII, pVIII, and pIX. According to former research, all five coat proteins can display polypeptides in the bacterial display system, but the most commonly used are pIII and pVIII proteins. Often, modification of coat proteins can improve the display of functional proteins, such as truncated protein pIII, using the CT domain.
Promoter is the core element of the expression vector. The most commonly used promoters of phagemid vectors include lactose promoter, arabinose promoter, alkaline phosphatase promoter, phage promoter, and T7 promoter. Usually, regulatable promoters are selected to facilitate the control of the expression of foreign proteins so as to prevent certain cell pressure caused by overexpression from affecting the expression of proteins.
3. Signal peptide
The phagemid vector usually selects the Sec-recognition signal peptide to complete the transport of display proteins, such as the pelB signal peptide. Holger Thie et al. proved that SRP-recognition signal peptide could be used for phage display, providing more space for signal selection. Nileena Velappan et al. showed that SEC-recognition signal peptide was suitable for displaying secreted proteins, such as antibodies; SRP- recognition signal peptide is suitable for transporting proteins that fold more rapidly, such as anchoring proteins. With the development of technology, more signal peptides will be used for phage display.
In addition to the above elements, the phagemid vector also includes a replication unit (ColEI) of E. coli plasmid, a marker for screening E. coli transformants (e.g., Amp, Kana), a restriction enzyme site for facilitating the insertion of foreign genes, a molecular tag for the detection and purification of the target protein...
Fig 3 The scheme of phagemid vector
Application of Phagemid Vector in Phage Display
Filamentous bacteriophages have five coat proteins. According to the different coat proteins contained in the phagemid vector, phagemids can be divided into types III, VIII, VI, VII, and IX. Because of the different molecular sizes and functions of each coat protein, the application of each type of phagemid vector is also different.
--Application of Yype III Phagemid Vector
The pIII protein molecule is 42 kDa, located at one end of the phage particle, with 3 to 5 copy numbers. It is a secondary structural protein that can fuse foreign proteins or peptides at the N or C terminal.
The most prominent advantage of the type III phagemid vector is that it has no strict restriction on the size of foreign peptides or proteins and can display larger proteins. However, the entry of phages into cells requires the interaction of sexual pili and coat protein pIII. When this type of phage is used, some cases will affect the infection of phage, and eventually lead to the loss of some clones and the reduction of the library size.
--Application of Type VIII Phagemid Vector
The pVIII protein molecule is 5.2 kDa, and the copy number is 2 700, which is the main structural protein. It is generally fused with foreign protein genes in the N-terminal, and there are also reports of peptides displayed in the C-terminal. In phage display system, the pVIII display system is usually used for random short peptides display.
The increased affinity demonstrated by this multivalent pVIII display is conducive to the screening of ligands with very low affinity. The disadvantage is that the number of peptides amino acids displayed is small, often limited to less than 6. Otherwise, it will affect the assembly of bacteriophage.
--Application of Type VI Phagemid Vector
Filamentous phage pVI has 112 amino acids and only 3 to 5 copies at the pIII protein's end, and pVI is only suitable for displaying foreign proteins at the Carboxyl terminal. Simon E-Hufton et al. constructed a pVI display system, successfully displaying CH3 of human IgG and alkaline phosphatase of Escherichia coli. This system was mainly used to construct cDNA phage surface display library and produced good results.
--Application of Type VII and Type IX Phagemid Vector
Filamentous phage pVII consists of 33 amino acids, and pIX consists of 32 amino acids, which are located in the tail of phage particles, opposite to pIII protein, with only 3 to 5 copies. pVII and pIX are only suitable for displaying foreign proteins at the amino-terminal.
pVII and PIX have successfully demonstrated foreign peptide and antibody libraries and achieved good screening results. Experiments have confirmed that pVII and pIX show that foreign proteins do not affect the infection ability of phages. pVII and pIX systems have a good application prospect for displaying bacteriophage antibody library.
Phagemid vector is an indispensable tool in phage display technology. According to different purposes and needs, researchers have continuously optimized various components of phagemids and constructed many phage vectors, which has greatly improved the level of phage display and expanded the application of phage display technology, providing an important means for obtaining peptides or antibodies valuable for cancer diagnosis and treatment. However, these phagemid vectors can only display a small part of the protein, the need for many special purposes, and timely monitoring of the display of multivalent foreign proteins; in short, there is still a need to continue to develop and modify the phagemid vectors. The transformation of phagemids should focus on the optimization of various components and the increase of protein display level and biological activity by increasing the library capacity and dual display system. It can be predicted that phagemids will play an important role in the fields of biosensors, molecular recognition, protein interaction, new drug development, molecular imaging, and molecular evolution.
The Features of Alpha Lifetech Inc.'s M13 Phage Display Library Construction Service
-- High throughput analysis of protein interactions
-- Excellent diversity: 108 - 1012
-- Display size: >110 kDa on pIII; <10 kDa on pVIII
-- Display density: 1 - 5 copies on pIII; <2700 copies on pVIII
-- Excellent for recombinant antibody production and antibody affinity maturation
-- Excellent for native species antibody library construction
Alpha Lifetech Inc. has mature M13 phage display system. Our high-quality libraries may achieve greater capacity, density, and proper orientation compared to ordinary display libraries, which furthers the examination of numerous protein candidates. If you are looking for a recombinant antibody production service or a peptide scanning library and would like more information on this program, please feel free to contact us.