Frequently Asked Questions
General Phage Display Questions
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What is Phage Display?
Phage display technology has been used for many purposes, especially in drug discovery for the development of therapeutic antibodies and peptides.
Phage display technology is a laboratory technique that employs genetically modified filamentous bacteriophages to display foreign variant peptides, antibodies, or other proteins. The phage particles are assembled into large and diverse phage display libraries. By linking each variant polypeptide sequence to its encoded DNA, binding to specific target molecules (such as biomarkers) can be rapidly assessed through a selection process known as biopanning.
During biopanning, the phage display library is exposed to the targets such as recombinant proteins, cells, or tissues in live animals. Unbound and non-specifically bound phages are then washed away while those bound via specific interactions remain strongly associated with the target. The bound phages can then be eluted using changes in pH, detergent, or by protease cleavage.
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What is a Phage Display Library?
A phage display library is a collection of bacteriophage viruses that have been genetically engineered to express a foreign polypeptide sequence that is fused to one of the viral coat proteins. The foreign sequence is most often a peptide or antibody that becomes displayed on the surface of the bacteriophage virus. A phage display library typically consists of 10^9-10^10 different phage clones, each displaying a different peptide or antibody. This enormous diversity of peptides or antibodies is one of the most powerful features of phage display technology.
Once a phage display library has been created, it can then be used in phage display selections to identify peptides or antibodies with desired properties such as binding to a specific target molecule (antigen). This is typically done through affinity selection techniques (biopanning), which involve iterative rounds of incubating the library with the target molecule. Unbound phage clones are removed by washing, whereas bound phages are collected by elution and subjected to the next round of affinity selection.
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What is Biopanning?
Biopanning of phage libraries is the most common method of selecting and identifying peptides and antibodies that bind to a target molecule. During biopanning, the phage display library is exposed to the target molecule and unbound and non-specifically bound phages are then washed away while those bound via specific interactions remain strongly associated with the target. The bound phages can then be eluted using changes in pH, detergent, or by protease cleavage.
Traditional biopanning procedures include the same overall key steps as shown below:
- Incubation of the phage display library with the target of interest (affinity selection).
- Removal of unbound phage by washing.
- Collection of bound phage by elution.
- Amplification of collected phage.
- Repetition of steps 1-4 for typically a total of four rounds.
- Identification of collected phage by DNA sequencing.
While phage display biopanning is a relatively simple and efficient technique, it requires careful optimization of several parameters in order to achieve the best results. These include the choice of the type of phage library, phage, and target molecule concentration, cell type (tumor cells, bacterial cells, etc.), incubation conditions such as temperature and time, as well as the amplification method of collected phage.
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What is a Phagemid?
Phage display technology is typically used in drug discovery for the high-throughput selection of peptides, antibodies, antibody fragments, and nanobodies. Phagemids are a type of commonly used vector in phage display technology.
Phagemids are plasmid-based vectors that contain both phage and bacterial origins of replication, and an antibiotic selection gene. Additionally, a phagemid vector typically encodes a coat protein (either pIII or pVIII) that is fused to a foreign DNA sequence to be used for the display of full-length antibodies, antibody fragments, nanobodies, or peptides. Since phagemid vectors are plasmid-based, they do not require the presence of helper phage proteins to replicate in E. coli. However, helper phages are essential for the production of phage clones since these contain essential genes that are required to assemble and release phage particles.
A major advantage of phagemids is the relatively small size of the vector DNA that enables easy transformation in E. coli. The ability to efficiently transform E. coli is directly related to the resulting diversity of the phage display library and is thus one of the most important considerations to make when choosing an appropriate vector for phage display.
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What is a Bacteriophage Vector?
Phage display technology was developed by Dr. George P. Smith in 1985 at the University of Missouri. He utilized the filamentous phage genome to create a bacteriophage vector that displays foreign peptide sequences on coat protein III (pIII). Today, two different types of vectors are typically used in phage display technology; phagemids and bacteriophage vectors, which are based on the original work by Dr. George Smith.
The filamentous bacteriophage vectors encode a modified version of the full phage genome. In addition to the wild-type genes, most bacteriophage vectors contain an antibiotic selection gene, as well as a recombinant hybrid gene encoding the foreign peptide or antibody fused to the coat protein. Expression of both the wild type and hybrid genes allows for lower levels of display of the foreign polypeptide. This is often an advantage as low valency often leads to the selection of high-affinity peptide sequences.
Bacteriophage vectors are typically not suitable for the display of antibodies and antibody fragments since their large size impairs the infection of E. coli and complicates transformation. However, bacteriophage vectors have been successfully used in peptide phage display. An advantage of bacteriophage vectors is that they do not require the use of helper phage for the propagation of phage particles as the genome contains all of the necessary genes for replication.
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What is a Helper Phage?
Helper phages are viruses used in the type of phage display technology that employs peptides or antibody libraries based on the use of a phagemid vector. Phagemids are plasmid-based vectors that typically encode a coat protein (either pIII or pVIII) that is fused to a foreign DNA sequence to be used for the display of full-length antibodies, antibody fragments, nanobodies, or peptides. When utilizing a phagemid-based phage display library, helper phages are essential for the production of phage clones. While the phagemid vector contains the DNA sequence for the genetically modified coat protein that is used for the display of the foreign peptide or antibody, the helper phage contains essential genes that are required to assemble and release phage particles. In short, the helper phage provides the essential machinery to package and replicate the genetic material so it can be presented on the viral surface.
Using helper phage offers several advantages. One is that the production of either the phagemid DNA or viral progeny can be controlled by the omission or addition of the helper phage to the culture conditions, respectively. This offers an additional advantage in that the production of soluble recombinant proteins encoded in the phagemid can be regulated.
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