Phage display
Encyclopedia
Phage display is a method for the study of protein–protein, protein–peptide, and protein–DNA interactions that uses bacteriophage
s to connect proteins with the genetic information that encodes them. Phage Display was originally invented by George P. Smith in 1985 and he demonstrated the display of peptides on filamentous phage by fusing the peptide of interest on to gene3 of filamentous phage. This technology was further developed and improved by groups at the MRC Laboratory of Molecular Biology with Winter and McCafferty and The Scripps Research Institute with Lerner and Barbas for display of proteins like antibodies for therapeutic protein engineering. The connection between genotype
and phenotype
enables large libraries of proteins to be screened and amplified in a process called in vitro
selection, which is analogous to natural selection
. The most common bacteriophages used in phage display are M13 and fd filamentous phage
, though T4
, T7
, and λ
phage have also been used.
into the pIII or pVIII gene, encoding either the minor or major coat protein, respectively. Multiple cloning site
s are sometimes used to ensure that the fragments are inserted in all three possible frames
so that the cDNA fragment is translated in the proper frame. The phage gene and insert DNA hybrid is then transformed into Escherichia coli (E. coli) bacterial cells such as TG1, SS320, ER2738, or XL1-Blue E. coli. If a "phagemid
" vector is used (a simplified display construct vector) phage particles will not be released from the E. coli cells until they are infected with helper phage
, which enables packaging of the phage DNA and assembly of the mature virions with the relevant protein fragment as part of their outer coat on either the minor (pIII) or major (pVIII) coat protein.
By immobilizing a relevant DNA or protein target(s) to the surface of a well, a phage that displays a protein that binds to one of those targets on its surface will remain while others are removed by washing. Those that remain can be eluted, used to produce more phage (by bacterial infection with helper phage) and so produce a phage mixture that is enriched with relevant (i.e. binding) phage. The repeated cycling of these steps is referred to as 'panning'
, in reference to the enrichment of a sample of gold by removing undesirable materials.
Phage eluted in the final step can be used to infect a suitable bacterial host, from which the phagemids can be collected and the relevant DNA sequence excised and sequenced to identify the relevant, interacting proteins or protein fragments.
The use of a helper phage can be eliminated by using 'bacterial packaging cell line' technology.
Phage display is also a widely used method for in vitro protein evolution (also called protein engineering
). As such, phage display is a useful tool in drug discovery
. It is used for finding new ligand
s (enzyme inhibitors, receptor agonists and antagonists) to target proteins.
Invention of antibody phage display by laboratories at the MRC Laboratory of Molecular Biology led by Greg Winter and John McCafferty
and at The Scripps Research Institute led by Richard Lerner and Carlos F. Barbas revolutionised antibody drug discovery. In 1991, The Scripps group reported the first display and selection of human antibodies on phage. This initial study described the rapid isolation of human antibody Fab fragements that bound tetanus toxin and the method was then extended to rapidly clone human anti-HIV-1 antibodies for vaccine design and therapy. Following the pioneering disclosures of these laboratories phage display of antibody libraries became a powerful method for both studying the immune response as well as a method to rapidly select and evolve human antibodies for therapy. Antibody phage display was later used by Carlos F. Barbas at The Scripps Research Institute to create the first synthetic human antibody libraries, thereby allowing human antibodies to be created in vitro from synthetic diversity elements. Antibody libraries displaying millions of different antibodies on phage are frequently used in the pharmaceutical industry for isolation of highly specific therapeutic antibody leads, for development into primarily anti-cancer or anti-inflammatory antibody drugs. One of the most successful was HUMIRA (adalimumab
), discovered by Cambridge Antibody Technology
as D2E7 and developed and marketed by Abbott Laboratories
. HUMIRA, an antibody to TNF alpha, was the world's first fully human antibody, which achieved annual sales exceeding $1bn.
Competing methods for in vitro protein evolution are yeast display
, bacterial display
, ribosome display
, and mRNA display
.
s have been an important part of phage display study . Databases , programs and web servers have been widely used to exclude target-unrelated peptides , characterize small molecules-protein interactions and map protein-protein interactions.
Bacteriophage
A bacteriophage is any one of a number of viruses that infect bacteria. They do this by injecting genetic material, which they carry enclosed in an outer protein capsid...
s to connect proteins with the genetic information that encodes them. Phage Display was originally invented by George P. Smith in 1985 and he demonstrated the display of peptides on filamentous phage by fusing the peptide of interest on to gene3 of filamentous phage. This technology was further developed and improved by groups at the MRC Laboratory of Molecular Biology with Winter and McCafferty and The Scripps Research Institute with Lerner and Barbas for display of proteins like antibodies for therapeutic protein engineering. The connection between genotype
Genotype
The genotype is the genetic makeup of a cell, an organism, or an individual usually with reference to a specific character under consideration...
and phenotype
Phenotype
A phenotype is an organism's observable characteristics or traits: such as its morphology, development, biochemical or physiological properties, behavior, and products of behavior...
enables large libraries of proteins to be screened and amplified in a process called in vitro
In vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
selection, which is analogous to natural selection
Natural selection
Natural selection is the nonrandom process by which biologic traits become either more or less common in a population as a function of differential reproduction of their bearers. It is a key mechanism of evolution....
. The most common bacteriophages used in phage display are M13 and fd filamentous phage
Filamentous phage
A filamentous phage is a type of bacteriophage shaped like a rod filament. Filamentous phages usually contain a genome of single-stranded DNA and infect Gram-negative bacteria.-Types of filamentous phage:*Ff phages - these infect E...
, though T4
Enterobacteria phage T4
Enterobacteria phage T4 is a bacteriophage that infects E. coli bacteria. Its DNA is 169–170 kbp long, and is held in an icosahedral head. T4 is a relatively large phage, at approximately 90 nm wide and 200 nm long...
, T7
T7 phage
Bacteriophage T7 is a bacteriophage capable of infecting susceptible bacterial cells. It infects most strains of Escherichia coli Bacteriophage T7 is a bacteriophage capable of infecting susceptible bacterial cells. It infects most strains of Escherichia coli Bacteriophage T7 is a bacteriophage...
, and λ
Lambda phage
Enterobacteria phage λ is a temperate bacteriophage that infects Escherichia coli.Lambda phage is a virus particle consisting of a head, containing double-stranded linear DNA as its genetic material, and a tail that can have tail fibers. The phage particle recognizes and binds to its host, E...
phage have also been used.
Principle
Like the two-hybrid system, phage display is used for the high-throughput screening of protein interactions. In the case of M13 filamentous phage display, the DNA encoding the protein or peptide of interest is ligatedDNA ligase
In molecular biology, DNA ligase is a specific type of enzyme, a ligase, that repairs single-stranded discontinuities in double stranded DNA molecules, in simple words strands that have double-strand break . Purified DNA ligase is used in gene cloning to join DNA molecules together...
into the pIII or pVIII gene, encoding either the minor or major coat protein, respectively. Multiple cloning site
Multiple cloning site
A multiple cloning site , also called a polylinker, is a short segment of DNA which contains many restriction sites - a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring only once within a given plasmid. MCSs are commonly used during...
s are sometimes used to ensure that the fragments are inserted in all three possible frames
Reading frame
In biology, a reading frame is a way of breaking a sequence of nucleotides in DNA or RNA into three letter codons which can be translated in amino acids. There are 3 possible reading frames in an mRNA strand: each reading frame corresponding to starting at a different alignment...
so that the cDNA fragment is translated in the proper frame. The phage gene and insert DNA hybrid is then transformed into Escherichia coli (E. coli) bacterial cells such as TG1, SS320, ER2738, or XL1-Blue E. coli. If a "phagemid
Phagemid
A phagemid or phasmid is a type of cloning vector developed as a hybrid of the filamentous phage M13 and plasmids to produce a vector that can grow as a plasmid, and also be packaged as single stranded DNA in viral particles...
" vector is used (a simplified display construct vector) phage particles will not be released from the E. coli cells until they are infected with helper phage
Helper virus
A helper virus is a virus used when producing copies of a helper dependent viral vector which does not have the ability to replicate on its own. The helper virus is used to coinfect cells alongside the viral vector and provides the necessary enzymes for replication of the genome of the viral vector....
, which enables packaging of the phage DNA and assembly of the mature virions with the relevant protein fragment as part of their outer coat on either the minor (pIII) or major (pVIII) coat protein.
By immobilizing a relevant DNA or protein target(s) to the surface of a well, a phage that displays a protein that binds to one of those targets on its surface will remain while others are removed by washing. Those that remain can be eluted, used to produce more phage (by bacterial infection with helper phage) and so produce a phage mixture that is enriched with relevant (i.e. binding) phage. The repeated cycling of these steps is referred to as 'panning'
Biopanning
Biopanning is an affinity selection technique which selects for peptides that bind to a given target . All peptide sequences obtained from biopanning using combinatorial peptide libraries have been stored in a special database with the name MimoDB , which is freely available...
, in reference to the enrichment of a sample of gold by removing undesirable materials.
Phage eluted in the final step can be used to infect a suitable bacterial host, from which the phagemids can be collected and the relevant DNA sequence excised and sequenced to identify the relevant, interacting proteins or protein fragments.
The use of a helper phage can be eliminated by using 'bacterial packaging cell line' technology.
General protocol
- Target proteins or DNA sequences are immobilised to the wells of a microtiter plateMicrotiter plateA Microtiter plate or microplate or microwell plate, is a flat plate with multiple "wells" used as small test tubes. The microplate has become a standard tool in analytical research and clinical diagnostic testing laboratories...
. - Many genetic sequences are expressed in a bacteriophageBacteriophageA bacteriophage is any one of a number of viruses that infect bacteria. They do this by injecting genetic material, which they carry enclosed in an outer protein capsid...
library in the form of fusions with the bacteriophage coat protein, so that they are displayed on the surface of the viral particle. The protein displayed corresponds to the genetic sequence within the phage. - This phage-display library is added to the dish and after allowing the phage time to bind, the dish is washed.
- Phage-displaying proteins that interact with the target molecules remain attached to the dish, while all others are washed away.
- Attached phage may be eluted and used to create more phage by infection of suitable bacterial hosts. The new phage constitutes an enriched mixture, containing considerably less irrelevant phage (i.e. non-binding) than were present in the initial mixture.
- The DNA within the interacting phage contains the sequences of interacting proteins, and following further bacterial-based amplification, can be sequenced to identify the relevant, interacting proteins or protein fragments.
Applications
The applications of this technology include determination of interaction partners of a protein (which would be used as the immobilised phage "bait" with a DNA library consisting of all coding sequences of a cell, tissue or organism) so that new functions or mechanisms of function of that protein may be inferred. The technique is also used to determine tumour antigens (for use in diagnosis and therapeutic targeting) and in searching for protein–DNA interactions using specially-constructed DNA libraries with randomised segments.Phage display is also a widely used method for in vitro protein evolution (also called protein engineering
Protein engineering
Protein engineering is the process of developing useful or valuable proteins. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles....
). As such, phage display is a useful tool in drug discovery
Drug discovery
In the fields of medicine, biotechnology and pharmacology, drug discovery is the process by which drugs are discovered or designed.In the past most drugs have been discovered either by identifying the active ingredient from traditional remedies or by serendipitous discovery...
. It is used for finding new ligand
Ligand
In coordination chemistry, a ligand is an ion or molecule that binds to a central metal atom to form a coordination complex. The bonding between metal and ligand generally involves formal donation of one or more of the ligand's electron pairs. The nature of metal-ligand bonding can range from...
s (enzyme inhibitors, receptor agonists and antagonists) to target proteins.
Invention of antibody phage display by laboratories at the MRC Laboratory of Molecular Biology led by Greg Winter and John McCafferty
John McCafferty
John McCafferty is a British scientist, one of the founders of Cambridge Antibody Technology, well known as one of the inventors of scFv antibody fragment phage display, a technology that revolutionised the monoclonal antibody drug discovery...
and at The Scripps Research Institute led by Richard Lerner and Carlos F. Barbas revolutionised antibody drug discovery. In 1991, The Scripps group reported the first display and selection of human antibodies on phage. This initial study described the rapid isolation of human antibody Fab fragements that bound tetanus toxin and the method was then extended to rapidly clone human anti-HIV-1 antibodies for vaccine design and therapy. Following the pioneering disclosures of these laboratories phage display of antibody libraries became a powerful method for both studying the immune response as well as a method to rapidly select and evolve human antibodies for therapy. Antibody phage display was later used by Carlos F. Barbas at The Scripps Research Institute to create the first synthetic human antibody libraries, thereby allowing human antibodies to be created in vitro from synthetic diversity elements. Antibody libraries displaying millions of different antibodies on phage are frequently used in the pharmaceutical industry for isolation of highly specific therapeutic antibody leads, for development into primarily anti-cancer or anti-inflammatory antibody drugs. One of the most successful was HUMIRA (adalimumab
Adalimumab
Adalimumab is the third TNF inhibitor, after infliximab and etanercept, to be approved in the United States. Like infliximab and etanercept, adalimumab binds to TNFα, preventing it from activating TNF receptors; adalimumab was constructed from a fully human monoclonal antibody, while infliximab...
), discovered by Cambridge Antibody Technology
Cambridge Antibody Technology
Cambridge Antibody Technology was a biotechnology company headquartered in Cambridge, United Kingdom...
as D2E7 and developed and marketed by Abbott Laboratories
Abbott Laboratories
Abbott Laboratories is an American-based global, diversified pharmaceuticals and health care products company. It has 90,000 employees and operates in over 130 countries. The company headquarters are in Abbott Park, North Chicago, Illinois. The company was founded by Chicago physician, Dr....
. HUMIRA, an antibody to TNF alpha, was the world's first fully human antibody, which achieved annual sales exceeding $1bn.
Competing methods for in vitro protein evolution are yeast display
Yeast display
Yeast display is a technique used in the field of protein engineering. The yeast display technique was first published by the laboratory of Professor K. Dane Wittrup. The technology was sold to Abbott Laboratories in 2001....
, bacterial display
Bacterial display
Bacterial display is a protein engineering technique used for in vitro protein evolution...
, ribosome display
Ribosome display
Ribosome display is a technique used to perform in vitro protein evolution to create proteins that can bind to a desired ligand. The process results in translated proteins that are associated with their mRNA progenitor which is used, as a complex, to bind to an immobilized ligand in a selection step...
, and mRNA display
MRNA display
mRNA display is a display technique used for in vitro protein, and/or peptide evolution to create molecules that can bind to a desired target. The process results in translated peptides or proteins that are associated with their mRNA progenitor via a puromycin linkage. The complex then binds to...
.
Bioinformatics Resources and Tools
Databases and computational tools for mimotopeMimotope
A mimotope is a macromolecule, often a peptide, which mimics the structure of an epitope. Because of this property it causes an antibody response similar to the one elicited by the epitope. An antibody for a given epitope antigen will recognize a mimotope which mimics that epitope. Mimotopes are...
s have been an important part of phage display study . Databases , programs and web servers have been widely used to exclude target-unrelated peptides , characterize small molecules-protein interactions and map protein-protein interactions.
See also
- Two-hybrid system, an alternative technique for studying protein–protein interactions
- protein–protein interactions