Phage display


Phage display is a laboratory technique for the study of protein interactions that uses bacteriophages to produce and "display" the proteins on their surfaces. Since the proteins remain attached to the surface of the phage, it is possible to isolate the phages displaying desirable proteins from among very large collections of phages, using e.g. other protein or DNA molecules as baits. The DNA of the selected phages can then be sequenced to establish the identity of selected proteins. The phages themselves can be further propagated in bacteria to amplify or diversify the selected protein library, with potential for conducting directed evolution experiments with multiple rounds of selection and diversification.
Specifically, a gene encoding a protein of interest is inserted into a phage coat protein gene, causing the phage to "display" the protein on its outside while containing the gene for the protein on its inside. This couples the genotype, phenotype in the context of an organism capable of replication. The phages displaying proteins of interest can then be selected using other proteins or DNA sequences in order to e.g., identify natural protein binding partners or antibodies with a high binding affinity.
The most common bacteriophages used in phage display are M13 and fd filamentous phage, though T4, T7, and λ phage have also been used.

History

Phage display was first described by George P. Smith in 1985, when he demonstrated the display of peptides on filamentous phage by fusing the virus's capsid protein to one peptide out of a collection of peptide sequences. This displayed the different peptides on the outer surfaces of the collection of viral clones, where the screening step of the process isolated the peptides with the highest binding affinity.
In 1988, Stephen Parmley and George Smith described biopanning for affinity selection and demonstrated that recursive rounds of selection could enrich for clones present at 1 in a billion or less. In 1990, Jamie Scott and George Smith described creation of large random peptide libraries displayed on filamentous phage.
Phage display technology was further developed and improved by groups at the Laboratory of Molecular Biology with Greg Winter and John McCafferty, The Scripps Research Institute with Richard Lerner and Carlos Barbas and the German Cancer Research Center with Frank Breitling and Stefan Dübel for display of proteins such as antibodies for therapeutic protein engineering.
Smith and Winter were awarded a half share of the 2018 Nobel Prize in chemistry for their contribution to developing phage display. A patent by George Pieczenik claiming priority from 1985 also describes the generation of peptide libraries.

Principle

In the case of M13 filamentous phage display, the DNA encoding the protein of interest is inserted into the gene encoding either the minor or the major coat protein. The modified coat protein gene and the rest of the phage genome is then introduced into E. coli bacteria, which produce phage virions with the relevant protein fragment as part of their outer coat phage and the DNA encoding for these proteins packaged inside the phage.
The phages can then be selected using e.g. DNA or protein molecules immobilized on the surface of a microplate. Specifically, phages that display proteins that binds to those targets will remain attached, while others will be removed by washing. Those that remain can be eluted and amplified by bacterial infection. The repeated cycling of selection, elution and amplification is sometimes 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 sequenced to identify the selected proteins.
During amplification step, additional mutations may be introduced into the genes encoding the proteins of interest, enabling a directed evolution protocol.
Elution can be done combining low-pH elution buffer with sonification, which, in addition to loosening the peptide-target interaction, also serves to detach the target molecule from the immobilization surface. This ultrasound-based method enables single-step selection of a high-affinity peptide.
The DNA encoding a fusion of coat protein and protein of interest is often encoded on a phagemid - a plasmid containing both a bacterial origin of replication and phage attachment sequence. This allows it to be maintained and amplified in bacteria without producing phage virons. When bacterial colony reaches a desired size, a helper plasmid is transformed into the bacteria to supply them with the rest of the phage genome, enabling viron production. Alternatively, these phage genes can maintained within bacteria under inducible promoters, obviating the need for separate helper plasmid introduction.

Applications

Applications of phage display technology include determination of interaction partners of a protein so that the function or the mechanism of the function of that protein may be determined. Phage display is also a widely used method for in vitro protein evolution. As such, phage display is a useful tool in drug discovery. It is used for finding new ligands to target proteins. The technique is also used to determine tumour antigens and in searching for protein-DNA interactions using specially-constructed DNA libraries with randomised segments. Recently, phage display has also been used in the context of cancer treatments - such as the adoptive cell transfer approach. In these cases, phage display is used to create and select synthetic antibodies that target tumour surface proteins. These are made into synthetic receptors for T-Cells collected from the patient that are used to combat the disease. Recently, M13 bacteriophages were genetically engineered to display an anti-GD2 single-chain variable fragment derived from the FDA-approved antibody Dinutuximab on their pIII coat protein. The engineered phages were subsequently loaded with hundreds of photosensitizer molecules to selectively deliver the payload to GD2-positive neuroblastoma cells. Upon activation by light or ultrasound, the photosensitizers induced targeted and precise killing of GD2-positive cells both in vitro and in vivo.
Competing methods for in vitro protein evolution include yeast display, bacterial display, ribosome display, and mRNA display.

Antibody maturation ''in vitro''

The invention of antibody phage display revolutionised antibody drug discovery. Initial work was done by laboratories at the MRC Laboratory of Molecular Biology, the Scripps Research Institute and the German Cancer Research Centre. 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 that bound tetanus toxin and the method was then extended to rapidly clone human anti-HIV-1 antibodies for vaccine design and therapy.
Phage display of antibody libraries has become 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 synthetic human antibody libraries, a principle first patented in 1990 by Breitling and coworkers, thereby allowing human antibodies to be created in vitro from synthetic diversity elements.
Antibody libraries displaying millions of different antibodies on phage are often used in the pharmaceutical industry to isolate highly specific therapeutic antibody leads, for development into antibody drugs primarily as anti-cancer or anti-inflammatory therapeutics. One of the most successful was adalimumab, discovered by Cambridge Antibody Technology as D2E7 and developed and marketed by Abbott Laboratories. Adalimumab, an antibody to TNF alpha, was the world's first fully human antibody to achieve annual sales exceeding $1bn.

General protocol

Below is the sequence of events that are followed in phage display screening to identify polypeptides that bind with high affinity to desired target protein or DNA sequence:
  1. Target proteins or DNA sequences are immobilized to the wells of a microtiter plate.
  2. Many genetic sequences are expressed in a bacteriophage 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.
  3. This phage-display library is added to the dish and after allowing the phage time to bind, the dish is washed.
  4. Phage-displaying proteins that interact with the target molecules remain attached to the dish, while all others are washed away.
  5. 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 than were present in the initial mixture.
  6. Steps 3 to 5 are optionally repeated one or more times, further enriching the phage library in binding proteins.
  7. Following further bacterial-based amplification, the DNA within the interacting phage is sequenced to identify the interacting proteins or protein fragments.

    Selection of the coat protein

Filamentous phages

pIII

pIII is the protein that determines the infectivity of the virion. pIII is composed of three domains connected by glycine-rich linkers. The N2 domain binds to the F pilus during virion infection freeing the N1 domain which then interacts with a TolA protein on the surface of the bacterium. Insertions within this protein are usually added in position 249, position 198 and at the N-terminus. However, when using the BamHI site located at position 198 one must be careful of the unpaired Cysteine residue that could cause problems during phage display if one is using a non-truncated version of pIII.
An advantage of using pIII rather than pVIII is that pIII allows for monovalent display when using a phagemid combined with a helper phage. Moreover, pIII allows for the insertion of larger protein sequences and is more tolerant to it than pVIII. However, using pIII as the fusion partner can lead to a decrease in phage infectivity leading to problems such as selection bias caused by difference in phage growth rate or even worse, the phage's inability to infect its host. Loss of phage infectivity can be avoided by using a phagemid plasmid and a helper phage so that the resultant phage contains both wild type and fusion pIII.
cDNA has also been analyzed using pIII via a two complementary leucine zippers system, Direct Interaction Rescue or by adding an 8-10 amino acid linker between the cDNA and pIII at the C-terminus.