Phage display is a laboratory technique for the study of protein–protein, protein–peptide, and protein–DNA interactions that uses bacteriophages to connect proteins with the genetic information that encodes them.[1] Phage Display was originally invented by Prof. George Pieczenik in 1983 USPTO disclosure document 118831 [[1]] cited as proof of date of conception in a continuation-in-part US Patent 5,866,363 originally filed in August 1985 [[2]] and told to Vidal de La Cruz by Prof. George Pieczenik when they met at NIH. Vidal de La Cruz was in turn credited for Professor Pieczenik's idea by George P. Smith in his 1985 publication, when he demonstrated the display of peptides on filamentous phage by fusing the peptide of interest on to gene III of filamentous phage.[1] This technology was further developed by Prof. George Pieczenik [[3]] reduced to practice 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 such as antibodies for therapeutic protein engineering. The most common bacteriophages used in phage display are variants of f1 discovered by Norton Zinder and Loeb [[4]].
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Contents
- 1 Principle
- 2 General protocol
- 3 Applications
- 4 Bioinformatics Resources and Tools
- 5 See also
- 6 References
- 7 Further reading
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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 ligated into the pIII or pVIII gene, encoding either the minor or major coat protein, respectively. This location can only accept small DNA fragments. Therefore, this vector was not used as a display phage vector until Prof. George Pieczenik re-introduced using f1 bacteriophage at its single HinD site in gene III (which does not exist in M13) as a vector for randomly synthesized DNA fragments. This created the first phage display library. This was done at the MRC Laboratory of Molecular Biology. George Smith only created a simple vector displaying a small DNA fragment of no consequence until the concept of inserting randomly generated sequences was introduced by Prof. George Pieczenik.
By immobilizing a relevant DNA or protein target(s) to the surface of a microtiter plate 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. Phage eluted in the final step can be used to infect a suitable bacterial host, plated on soft agar, and then the plaques can be picked and the DNA corresponding to protein that bound can be sequenced directly.
General protocol
- Target proteins or DNA sequences are immobilised 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 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.[2] The technique is also used to determine tumour antigens (for use in diagnosis and therapeutic targeting)[3] and in searching for protein-DNA interactions[4] 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). As such, phage display is a useful tool in drug discovery. It is used for finding new ligands (enzyme inhibitors, receptor agonists and antagonists) to target proteins.[5][6][7]
The invention of antibody phage display by Prof. George Pieczenik and reduced to practice by the technical staff using Zinder's f1 bacteriophage and Prof.Pieczenik's protocols and Cadbury reagents 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.[8][9] In 1991, The Scripps group reported the first display and selection of human antibodies on phage.[10] 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.[11][12][13][14][15] 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.[16][17][18][19] Antibody libraries displaying billions 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,[20] which achieved annual sales exceeding $1bn.[21] Prof. George Pieczenik priority claims 37 USC 102(e) are validated by Nobel Laureate Bruce Merrifield [[5]],[[6]]. Prof. George Pieczenik has sued many infringers and will sue Wikipedia if it continues to insist on publishing fraudulant inventorship claims when its editors have no understanding of what a continuation-in-part patent application is and that the submission date refers back to the original filing. The stupidity of the Wiki editors has created alot of noise and very little accurate information. They are the dupes of the pharma corporations and paid to publish pharma propaganda.
Competing methods for in vitro protein evolution are yeast display, bacterial display, ribosome display, and mRNA display.
Bioinformatics Resources and Tools
Databases and computational tools for mimotopes have been an important part of phage display study.[22] Databases,[23] programs and web servers have been widely used to exclude target-unrelated peptides,[24] 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
References
- ^ a b Smith GP (1985). "Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface". Science 228 (4705): 1315–1317. doi:10.1126/science.4001944. PMID 4001944.
- ^ Explanation of "Protein interaction mapping" from The Wellcome Trust
- ^ Hufton SE, Moerkerk PT, Meulemans EV, de Bruïne A, Arends JW, Hoogenboom HR (1999). "Phage display of cDNA repertoires: the pVI display system and its applications for the selection of immunogenic ligands". J. Immunol. Methods 231 (1-2): 39–51. doi:10.1016/S0022-1759(99)00139-8. PMID 10648926. http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T2Y-418YGFH-5&_user=1543454&_coverDate=12%2F10%2F1999&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000053633&_version=1&_urlVersion=0&_userid=1543454&md5=ad85a4991cf0cee17bfec69bc90dd707.
- ^ Gommans WM, Haisma HJ, Rots MG (2005). "Engineering zinc finger protein transcription factors: the therapeutic relevance of switching endogenous gene expression on or off at command". J. Mol. Biol. 354 (3): 507–19. doi:10.1016/j.jmb.2005.06.082. PMID 16253273. http://www.rug.nl/farmacie/onderzoek/basisEenheden/THErapeuticGeneModulation/publicaties/publicaties2004/2005_6.pdf?as=binary.
- ^ Lunder M, Bratkovic T, Doljak B, Kreft S, Urleb U, Strukelj B, Plazar N. (2005). "Comparison of bacterial and phage display peptide libraries in search of target-binding motif". Appl. Biochem. Biotechnol. 127 (2): 125–31. doi:10.1385/ABAB:127:2:125. PMID 16258189.
- ^ Bratkovic T, Lunder M, Popovic T, Kreft S, Turk B, Strukelj B, Urleb U. (2005). "Affinity selection to papain yields potent peptide inhibitors of cathepsins L, B, H, and K". Biochem. Biophys. Res. Commun. 332 (3): 897–903. doi:10.1016/j.bbrc.2005.05.028. PMID 15913550.
- ^ Lunder M, Bratkovic T, Kreft S, Strukelj B (2005). "Peptide inhibitor of pancreatic lipase selected by phage display using different elution strategies". J. Lipid Res. 2005 46 (7): 1512–6. doi:10.1194/jlr.M500048-JLR200. PMID 15863836.
- ^ McCafferty J, Griffiths A.D, Winter G, Chiswell D.J (1990). "Phage antibodies: filamentous phage displaying antibody variable domains". Nature 348 (63017): 552–554. Bibcode 1990Natur.348..552M. doi:10.1038/348552a0. PMID 2247164.
- ^ “Phage Display: A Laboratory Manual” C.F. Barbas, III, D.R. Burton, J.K. Scott, G.J. Silverman, Eds. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York; 2001; 736 pages.
- ^ “Assembly of combinatorial antibody libraries on phage surfaces: The gene III site” C.F. Barbas III, A.S. Kang, R.A. Lerner, and S.J. BenkovicProc. Natl. Acad. Sci. USA; 1991; 88(18); 7978-7982. doi:10.1073/pnas.88.18.7978
- ^ “A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals” D.R. Burton, C.F. Barbas III, M.A.A. Persson, S. Koenig, R.M. Chanock, and R.A. Lerner Proc. Natl. Acad. Sci. USA; 1991; 88(22); 10134-10137. doi:10.1073/pnas.88.22.10134
- ^ “Recombinant Human Fab fragments neutralize human type 1 immunodeficiency virus in vitro” C.F. Barbas III, E. Bjorling, F. Chiodi, N. Dunlop, D. Cababa, T.M. Jones, S.L. Zebedee, M.A.A. Persson, P.L. Nara, E. Norrby, and D.R. Burton Proc. Natl. Acad. Sci. USA; 1992; 89(19); 9339-9343. doi: 10.1073/pnas.89.19.9339
- ^ “Efficient Neutralization of Primary Isolates of HIV-1 by a Recombinant Human Monoclonal Antibody” D.R. Burton, J. Pyati, R. Koduri, S.J. Sharp, G.B. Thornton, P.W.H.I. Parren, L.S.W. Sawyer, M.R. Hendry, N. Dunlop, P.L. Nara, M. Lamacchia, E. Garratty, E.R. Stiehm, Y.J. Bryson, Y. Cao, J.P. Moore, D.D. Ho, and C.F. Barbas III Science; 1994; 266(5187); 1024-1026. doi: 10.1126/science.7973652
- ^ “CDR Walking Mutagenesis for the Affinity Maturation of a Potent Human anti-HIV-1 Antibody into the Picomolar RangeZ” W.-P. Yang, K. Green, S. Pinz-Sweeney, A.T. Briones, D.R. Burton, and C.F. Barbas III J. Mol. Biol.; 1995; 254; 392-403. doi:10.1006/jmbi.1995.0626
- ^ “In vitro evolution of a neutralizing human antibody to HIV-I to enhance affinity and broaden strain cross-reactivity” C.F. Barbas III, D. Hu, N. Dunlop, L. Sawyer, D. Cababa, R.M. Hendry, P.L. Nara, and D.R. Burton Proc. Natl. Acad. Sci. USA; 1994 91; 3809-3813.
- ^ “Semi-synthetic combinatorial antibody libraries: A chemical solution to the diversity problem” C.F. Barbas III, J.D. Bain, D.M. Hoekstra, and R.A. Lerner Proc. Natl. Acad. Sci. USA; 1992; 89(10); 4457-4461.
- ^ “High Affinity Self-Reactive Human Antibodies by Design and Selection: Targeting the Integrin Ligand Binding Site” C.F. Barbas III, L.R. Languino, and J.W. Smith Proc. Natl. Acad. Sci. USA; 1993; 90(21); 10003-10007. doi: 10.1073/pnas.90.21.10003
- ^ “Synthetic Human Antibodies: Selecting and Evolving Functional Proteins” C.F. Barbas III and J. Wagner Methods, A Companion to Methods in Enzymology; 1995; 8(2); 94-103. doi:10.1006/meth.1995.9997
- ^ “Synthetic Human Antibodies” C.F. Barbas III Nature Medicine; 1995; 1; 837-839. doi:10.1038/nm0895-837
- ^ Access : Billion dollar babies|[mdash]|biotech drugs as blockbusters : Nature Biotechnology
- ^ Cambridge Antibody: Sales update | Company Announcements | Telegraph
- ^ Huang, J; Ru, B, Dai, P (2011-01-18). "Bioinformatics resources and tools for phage display.". Molecules (Basel, Switzerland) 16 (1): 694–709. doi:10.3390/molecules16010694. PMID 21245805.
- ^ Huang, J; Ru, B, Zhu, P, Nie, F, Yang, J, Wang, X, Dai, P, Lin, H, Guo, FB, Rao, N (2011-11-03). "MimoDB 2.0: a mimotope database and beyond.". Nucleic Acids Research 40 (1): D271–7. doi:10.1093/nar/gkr922. PMID 22053087.
- ^ Huang, J; Ru, B, Li, S, Lin, H, Guo, FB (2010). "SAROTUP: scanner and reporter of target-unrelated peptides.". Journal of biomedicine & biotechnology 2010: 101932. doi:10.1155/2010/101932. PMC 2842971. PMID 20339521. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=2842971.
Further reading
- The ETH-2 human antibody phage library
- Sidhu, S. S.; Lowman, H. B.; Cunningham, B. C.; Wells, J. A. (2000). "Phage display for selection of novel binding peptides". Methods Enzymol 328: 333–363. doi:10.1016/S0076-6879(00)28406-1. PMID 11075354.
- Selection Versus Design in Chemical Engineering[dead link]
- McCafferty, J.; Griffiths, A.D.; Winter, G.; Chiswell, D.J. (1990). "Phage antibodies: filamentous phage displaying antibody variable domains". Nature 348 (6301): 552−554. Bibcode 1990Natur.348..552M. doi:10.1038/348552a0. PMID 2247164.
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Proteins: key methods of study
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| Experimental |
Protein purification · Green fluorescent protein · Western blot · Protein immunostaining · Protein sequencing · Gel electrophoresis/Protein electrophoresis · Protein immunoprecipitation · Peptide mass fingerprinting · Dual polarization interferometry · Microscale thermophoresis · Chromatin immunoprecipitation
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| Bioinformatics |
Protein structure prediction · Protein–protein docking · Protein structural alignment · Protein ontology · Protein–protein interaction prediction
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| Assay |
Enzyme assay · Protein assay · Secretion assay
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| Display techniques |
Bacterial display · mRNA display · Phage display · Ribosome display · Yeast display
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| Super resolution microscopy |
Vertico SMI
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