Phage Display FAQs

2018-12-03 Hits(995)

Q1: What is phage display?

A1: 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.

 

Q2: What are the advantages of phage display screening over other antibody development method?

A2: 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 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.

 

Q3: What are the difference among M13, T7, and T4 displaying system?

A3: 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.

 

Q4: Can I use M13 phage display system to construct a cDNA library?

A4: 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.

 

Q5: What media can be used to culture the phage?

A5: 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.

 

Q6: 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?

A6: 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 can not infect them.

 

Q7: Is there any antibiotic resistance for E. coli TG1 host strain?

A7: E. coli TG1 strain does not contain antibiotic resistance, while the phage-infected TG1 strain can be selected by 2YT-AK media (2YT containing 100 μg/mL ampicillin, 50 μg/mL kanamycin).

 

Q8: What is the difference between pfu and cfu?

A8: 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).

 

Q9: How do you know the constructed library is perfect?

A9: The affinity of Abs selected from an immune library is proportional to the size of the library, the larger of the library repertoire, the better of the antibody affinity. Usually, the size of the final library should reach 108. It is large enough for isolating high-affinity antibodies. Furthermore, as a perfect library, it should have a high diversity. The QC results of the final library showed that no common sequence was found from all of the randomly picked clones, which indicated that the diversity of the library was at a really high level.