Phage display
This article may be too technical for most readers to understand.(October 2018) |
Phage display is a laboratory technique for the study of
The most common bacteriophages used in phage display are
phage have also been used.History
Phage display was first described by
Principle
Like the
The use of a helper phage can be eliminated by using 'bacterial packaging cell line' technology.[9]
Elution can be done combining low-pH elution
Applications
Applications of phage display 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
Competing methods for in vitro protein evolution include yeast display, bacterial display, ribosome display, and mRNA display.[citation needed]
Antibody maturation in vitro
The invention of
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 (Patent CA 2035384), thereby allowing human antibodies to be created in vitro from synthetic diversity elements.[28][29][30][31]
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
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:[citation needed]
- Target proteins or DNA sequences are immobilized to the wells of a microtiter plate.
- 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.
- 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 elutedand 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.
- Steps 3 to 5 are optionally repeated one or more times, further enriching the phage library in binding proteins.
- 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 (N1, N2 and CT) connected by glycine-rich linkers.[34] 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.[34] Insertions within this protein are usually added in position 249 (within a linker region between CT and N2), position 198 (within the N2 domain) and at the N-terminus (inserted between the N-terminal secretion sequence and the N-terminus of pIII).[34] However, when using the BamHI site located at position 198 one must be careful of the unpaired Cysteine residue (C201) that could cause problems during phage display if one is using a non-truncated version of pIII.[34]
An advantage of using pIII rather than pVIII is that pIII allows for monovalent display when using a phagemid (plasmid derived from Ff phages) combined with a helper phage. Moreover, pIII allows for the insertion of larger protein sequences (>100 amino acids)[35] 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[36] or even worse, the phage's inability to infect its host.[34] 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.[34]
cDNA has also been analyzed using pIII via a two complementary leucine zippers system,[37] Direct Interaction Rescue[38] or by adding an 8-10 amino acid linker between the cDNA and pIII at the C-terminus.[39]
pVIII
pVIII is the main coat protein of Ff phages. Peptides are usually fused to the N-terminus of pVIII.[34] Usually peptides that can be fused to pVIII are 6-8 amino acids long.[34] The size restriction seems to have less to do with structural impediment caused by the added section[40] and more to do with the size exclusion caused by pIV during coat protein export.[40] Since there are around 2700 copies of the protein on a typical phages, it is more likely that the protein of interest will be expressed polyvalently even if a phagemid is used.[34] This makes the use of this protein unfavorable for the discovery of high affinity binding partners.[34]
To overcome the size problem of pVIII, artificial coat proteins have been designed.[41] An example is Weiss and Sidhu's inverted artificial coat protein (ACP) which allows the display of large proteins at the C-terminus.[41] The ACP's could display a protein of 20kDa, however, only at low levels (mostly only monovalently).[41]
pVI
pVI has been widely used for the display of cDNA libraries.[34] The display of cDNA libraries via phage display is an attractive alternative to the yeast-2-hybrid method for the discovery of interacting proteins and peptides due to its high throughput capability.[34] pVI has been used preferentially to pVIII and pIII for the expression of cDNA libraries because one can add the protein of interest to the C-terminus of pVI without greatly affecting pVI's role in phage assembly. This means that the stop codon in the cDNA is no longer an issue.[42] However, phage display of cDNA is always limited by the inability of most prokaryotes in producing post-translational modifications present in eukaryotic cells or by the misfolding of multi-domain proteins.
While pVI has been useful for the analysis of cDNA libraries, pIII and pVIII remain the most utilized coat proteins for phage display.[34]
pVII and pIX
In an experiment in 1995, display of Glutathione S-transferase was attempted on both pVII and pIX and failed.[43] However, phage display of this protein was completed successfully after the addition of a periplasmic signal sequence (pelB or ompA) on the N-terminus.[44] In a recent study, it has been shown that AviTag, FLAG and His could be displayed on pVII without the need of a signal sequence. Then the expression of single chain Fv's (scFv), and single chain T cell receptors (scTCR) were expressed both with and without the signal sequence.[45]
PelB (an amino acid signal sequence that targets the protein to the periplasm where a signal peptidase then cleaves off PelB) improved the phage display level when compared to pVII and pIX fusions without the signal sequence. However, this led to the incorporation of more helper phage genomes rather than phagemid genomes. In all cases, phage display levels were lower than using pIII fusion. However, lower display might be more favorable for the selection of binders due to lower display being closer to true monovalent display. In five out of six occasions, pVII and pIX fusions without pelB was more efficient than pIII fusions in affinity selection assays. The paper even goes on to state that pVII and pIX display platforms may outperform pIII in the long run.[45]
The use of pVII and pIX instead of pIII might also be an advantage because virion rescue may be undertaken without breaking the virion-antigen bond if the pIII used is wild type. Instead, one could cleave in a section between the bead and the antigen to elute. Since the pIII is intact it does not matter whether the antigen remains bound to the phage.[45]
T7 phages
The issue of using Ff phages for phage display is that they require the protein of interest to be translocated across the bacterial inner membrane before they are assembled into the phage.[46] Some proteins cannot undergo this process and therefore cannot be displayed on the surface of Ff phages. In these cases, T7 phage display is used instead.[46] In T7 phage display, the protein to be displayed is attached to the C-terminus of the gene 10 capsid protein of T7.[46]
The disadvantage of using T7 is that the size of the protein that can be expressed on the surface is limited to shorter peptides because large changes to the T7 genome cannot be accommodated like it is in M13 where the phage just makes its coat longer to fit the larger genome within it. However, it can be useful for the production of a large protein library for scFV selection where the scFV is expressed on an M13 phage and the antigens are expressed on the surface of the T7 phage.[47]
Bioinformatics resources and tools
Databases and computational tools for mimotopes have been an important part of phage display study.[48] Databases,[49] programs and web servers[50] have been widely used to exclude target-unrelated peptides,[51] characterize small molecules-protein interactions and map protein-protein interactions. Users can use three dimensional structure of a protein and the peptides selected from phage display experiment to map conformational epitopes. Some of the fast and efficient computational methods are available online.[50]
See also
- Directed evolution
- protein–protein interactions
- PelB leader sequence
Competing techniques:
- Two-hybrid system
- mRNA display
- Ribosome display
- Yeast display
References
- ^ PMID 4001944.
- PMID 11848876.
- PMID 16277371.
- PMID 12051907.
- PMID 3149606.
- PMID 1696028.
- ^ "The Nobel Prize in Chemistry 2018". NobelPrize.org. Retrieved 2018-10-03.
- ^ US patent 5866363, Pieczenik G, "Method and means for sorting and identifying biological information", published 1999-02-02
- PMID 17088290.
- PMID 18533899.
- ^ Explanation of "Protein interaction mapping" from The Wellcome Trust
- S2CID 45243314.
- PMID 15913550.
- PMID 15863836.
- PMID 10648926.
- PMID 16253273.
- ^ a b "CAR T Cells: Engineering Patients' Immune Cells to Treat Their Cancers". National Cancer Institute. 2013-12-06. Retrieved 9 February 2018.
- PMID 25629004.
- S2CID 4258014.
- ISBN 978-0-87969-740-2.
- PMID 1916287.
- PMID 1896445.
- PMID 1719545.
- PMID 1384050.
- PMID 7973652.
- PMID 7490758.
- PMID 8170992.
- PMID 1584777.
- PMID 7694276.
- .
- S2CID 6983649.
- S2CID 205266758.
- ^ Cambridge Antibody: Sales update | Company Announcements | Telegraph
- ^ ISBN 978-0-19-963873-4.
- PMID 10669603.
- PMID 20583018.
- PMID 7957259.
- PMID 7838733.
- S2CID 23009887.
- ^ S2CID 19331069.
- ^ PMID 10864510.
- S2CID 6171262.
- PMID 7616570.
- PMID 10339535.
- ^ PMID 21390283.
- ^ PMID 11606722.
- PMID 11687245.
- PMID 21245805.
- PMID 22053087.
- ^ PMID 20140073.
- PMID 20339521.
Further reading
- Ledsgaard L, Kilstrup M, Karatt-Vellatt A, McCafferty J, Laustsen AH (2018). "Basics of antibody phage display technology" (PDF). Toxins. 10 (6): 236. PMID 29890762.
- Selection Versus Design in Chemical Engineering
- The ETH-2 human antibody phage library Archived 2011-07-15 at the Wayback Machine
- Sidhu SS, Lowman HB, Cunningham BC, Wells JA (2000). "Phage display for selection of novel binding peptides". Applications of Chimeric Genes and Hybrid Proteins - Part C: Protein-Protein Interactions and Genomics. Methods in Enzymology. Vol. 328. pp. 333–63. )