Q: What is phage display? A: Phage display is a selection technique in which a library of variants of a peptide or protein is expressed on the outside of a phage virion, while the genetic material encoding each variant resides on the inside. This creates a physical linkage between each variant protein sequence and the DNA encoding it, which allows rapid partitioning based on binding affinity to a given target molecule (antibodies, enzymes, cell-surface receptors, etc.) by an in vitro selection process called "biopanning". Biopanning is carried out by incubating the pool of phage-displayed variants with a target of interest that has been immobilized on a plate or bead, washing away unbound phage, and eluting specifically bound phage. The eluted phage is then amplified in vivo and the process repeated, resulting in stepwise enrichment of the phage pool in favor of the tightest binding sequences. After 3-4 rounds of selection/amplification, individual clones are characterized by DNA sequencing.

Q: What are the advantages of phage display screening over other antibody development method? A: In comparison with hybridoma technology, phage display technology offers great advantages. Hybridoma method is only available in mouse, rat, hamster and guinea pig. On the other hand, phage display could be used to generate high-affinity monoclonal antibodies from all popular antibody production species, including but not limited to human, mouse, rat, rabbit, chicken, llama, camel, alpaca, cow, dog, sheep, monkey, and shark. Hybridoma-based monoclonal antibody development can only generate a small number of antibody candidates against a particular immunogen at a time; whereas phage display technology can present the entire antibody repertoire (e.g. 108) of an immunized animal, in which almost 10% of the antibodies are immunogen(s)-specific. With such a huge pool of potential binders, the chance is much better to use phage display technology to discover antibodies of the desired properties. In addition, using hybridoma technology, it is hard to incorporate an enriching step that can selectively isolate antibodies with the desired functionality. In most cases, all the hybridoma clones are produced first and then validated one by one. In contrast, phage display technology allows various enriching strategies: antibodies of desired properties can be enriched thus those without the desired functionality can be excluded from further validation. For example, antibody library screening can be done using the target to capture strong binders while using the controls to block/deplete cross-reactive binders. In summary, this immune antibody library approach allows collecting almost all antibodies in the animals and isolating the strongest binders from the collection. Moreover, counter-selection can be easily incorporated.

Q: What are the difference among M13, T7, and T4 displaying system? A: M13 filamentous phage has always been the most popular option and extensively used in various types of research, among many other types of phages. The viral coat consists of five different capsid proteins, including one major capsid pVIII (2,700 copies) and four minor capsids (pIII and pVI at one end while pVII and pIX at the other end). Unlike T4 and T7, M13 is lysogenic phage which is assembled in the periplasm and secreted out of the bacterial membrane without lysing the host. The multiple capsid proteins on M13 phage allow the comprehensive display choices for a variety of peptides and proteins with distinctive characteristics. All five capsids have been successfully used for foreign domain display with unique vectors. pIII and pVIII are mostly the preferred choices for M13 phage display. Bacteriophage T4 is distinct from M13 in many aspects. T4 has much larger size, tailed structure and double-stranded DNA (dsDNA) genome encoding 50 different proteins. Larger genome DNA allows larger insertions of foreign proteins. Two different domains could be displayed on HOC and SOC, the two non-essential coat proteins. Both N- and C-terminal insertion are available. Without membrane secreting process, host toxicity and confirmation changes could be avoided using T4 phage. T7 phages have a shorter lifecycle compared to filamentous phages and lambda phages. The assembly of the progeny phages are in bacterial cytoplasm and released by lysis of cell envelope, therefore there is no size limitation and host toxicity resulting from the secreting process. In addition, T7 phages are very stable at extreme conditions where other phages could not survive.

Q: Can I use M13 phage display system to construct a cDNA library? A: Usually, M13 is not suitable for cDNA expression, because the in-frame expression between the leader sequence (required for secretion) and the N-terminus of coat protein pIII or pVIII is required for M13 phage display. In order to fuse the corresponding protein to the coat protein properly, an insert must be in the correct reading frame at both ends and contains no in-frame stop codons. This results in a vanishingly small number of productive clones in M13 cDNA libraries.

Q: What are the differences between pIII and pVIII display? A: Minor coat protein pIII and the major coat protein pVIII proved most effective for the display of proteins and peptides. pIII could incorporate and present larger inserts compared to pVIII, it is the scaffold of choice in most cases of ORF and protein display. There are 5 copies of pIII protein on the phage virion. Both short peptides and large protein could be fused to pIII. The fusion protein will be present at low valency, 1-5 copies, therefore beneficial in selecting high-affinity binders. pVIII (encoded by genes VIII) is the major structural protein of the virus. Approximately 2,800 copies of pVIII are required to coat one full-length wild-type virion. Therefore, pVIII has high copy number and multivalent display. Multivalent display reduces selection for this property, by greatly increasing avidity (effective affinity) to the point where weak and strong ligands cannot be distinguished. On the other hand, multivalency could be a distinct advantage in other applications. Such is the case when it is desirable to accumulate a broad spectrum of peptides as potential leads; the identified peptides can be ordered subsequently in accordance with their affinity and selectivity using monovalent display. In pVIII display systems, there is a sharp limit on the length of the displayed peptide. The pVIII display systems will only accommodate very short peptides, ~100 amino acids, especially in high DNA copy number systems. the pVIII display system is able to display 300-500 copies of the pVIII fusion peptide/protein. Creative Biolabs is an expert on both pIII and pVIII display systems, we’ll work closely with you to select the optimal system for your projects.

Q: Do I need an import permit for a phage? A: No, phages are considered non-pathogenic to humans, animals or plants.

Q: What media can be used to culture the phage? A: TSB/TSA (trypticase soy broth/trypticase soy agar) can be used to grow most of the phages. However, the appropriate media can be various due to different phage display systems.

Q: What is the molecular weight of the coat protein pIII? A: For the unprocessed pIII (with a leader sequence but no displayed proteins or peptides), the molecular weight is 44,651 Da; for the mature pIII (without the leader sequence), it is 42,579 Da. However, pIII usually shows an apparent molecular weight of 60-65 kDa by SDS-PAGE, due to the unusual glycine-rich spacer regions between the domains of the protein.

Q: Could I display antibody fragments or proteins at the N-terminus of coat protein pIII with M13KE? A: M13KE is not amenable to antibody or protein expression, because in this system each copy of pIII displays the encoded peptide sequence. Any insert that interferes with phage particle assembly or pIII function will not make viable phage. We consider 25-50 amino acids to be the maximum range for this system.

Q: What kinds of E. coli strains can be infected by bacteriophage M13? Does it have the chance to contaminate all cell strains in our laboratory? A: The M13 phage is a kind of filamentous bacteriophage, which uses the tip of the bacterial F conjugative pilus as a receptor to facilitate the infection process. Thus, they are only specific for E. coli strains containing the F plasmid (F+). For your strains in your lab, we suggest you check whether they contain the F plasmid. If they don’t, the M13 phage cannot infect them.

Q: Is the phagemid vector used for the construction of custom libraries available? A: The vectors used for constructing all phage display libraries are available for sale, for research use only. However, the full sequence of the vector is private, we can only provide the schematic map, the primer, and the restriction enzyme information to you for PCR and DNA sequencing.

Q: Is there any antibiotic resistance for E. coli TG1 host strain? A: E. coli TG1 strain does not contain antibiotic resistance, while the phage-infected TG1 strain can be selected by 2YT-AK media.

Q: Could the helper phage be titrated by blue-white screening? A: The helper phage provided by Creative Biolabs does not contain lacZα but carry the KanR gene for antibiotic selection, thus cannot be distinguished through blue-white screening.

Q: What is the difference between pfu and cfu? A: The pfu, refers to the plaque-forming unit, is a measure of the quantity of individual infectious particles, and is usually used to count bacteriophage. The cfu, refers to the colony-forming unit, is a measure of viable cells in which a colony represents an aggregate of cells derived from a single progenitor cell, and is usually used to count bacteria (e.g. E. coli).

Q: How many clones are provided following binding/screening tests? Are these clones sequenced? A: In the clone validation stage, 40-300 clones will usually be tested (more clones could be validated upon request). We can perform phage ELISA, DNA sequencing, and soluble protein ELISA to confirm the activity. Other validation tests can also be performed upon request. Usually > 3-10 unique clones could be discovered. Our customer can be the sole owner of these identified clones. At this time, we are proud to offer an advanced high-throughput antibody discovery platform, Magic™ Therapeutic Antibody Discovery Platform (MTADP). After the library screening, MADTP could identify all the unique antibody sequences, with VH-VL pairing information, in the enriched library (~105). While using ELISA, only a very small portion, 40-300 clones, will be analyzed. It's very important to have a large number of antibody candidates if we want to obtain good therapeutic/diagnostic antibodies. Therefore, MTADP is highly recommended as there is a much better chance to identify functional antibodies from a large pool of antibody candidates.

Q: Can we ask supernatant or a small amount of purified antibodies for internal test? A: After the screening, a number of random clones will be validated. The supernatant of the positive clones can be provided to the customers as an additional deliverable. We also provide purified scFv/Fab/IgG production services upon request.

Q: Are anti-M13 antibodies available? A: Yes, we can offer the anti-M13 antibodies. We can offer the Recombinant Anti-M13 Major Coat Protein Antibody (CBMAB-0206MC) or other custom anti-M13 antibodies of your choice. Please contact our customer service for more details.