Biological Sciences
Phage Display
Phage display is a laboratory technique used to study protein-protein interactions and identify specific peptide sequences that bind to a target molecule. It involves genetically engineering bacteriophages to display foreign peptides on their surface, allowing researchers to screen and select for peptides with desired binding properties. This method has applications in drug discovery, vaccine development, and understanding protein function.
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12 Key excerpts on "Phage Display"
eBook - ePub- Boriana Marintcheva(Author)
- 2017(Publication Date)
- Academic Press(Publisher)
Chapter 5 Phage Display Abstract Phage Display is a powerful technique for studying protein–ligand interactions most frequently applied to protein–protein, protein–peptide, and protein–nucleic acids interactions. The genetic code for the protein/peptide of interest is inserted in the genome of a phage and subsequently “displayed” on the surface of the viral particle as a fusion to natural coat protein. Libraries of protein/peptide variants are tested against ligand(s) of interest. Proteins/peptides binding to the specific target are selected by 3–5 rounds of affinity-driven biopanning and subsequently identified by sequencing the genome of the phages displaying them. Phage Display is widely used for a selection of proteins/peptides with desired binding properties for the purpose of a broad array of therapeutic, research, and nanotechnology-related applications. Keywords Biopanning; Peptide library; Phage Display; Phage-assisted continuous evolution Phage Display is a well-established technique for identification, selection, and evolution of protein–ligand interactions widely used to address basic science questions, as well as to develop top-notch technology in diverse fields ranging from research tools to personalized medicine and nanotechnology. Phage Display has more than 30 years long history that has reaffirmed the power of the approach and resulted in the development of multiple display techniques to meet the continuous challenge of studying the vast diversity of protein–ligand interactions taking place in living matter. The first reports describing Phage Display were published in the 1980s and ever since the technique has been continuously improving as advances in molecular biology and various technologies are being used to build on its simple but brilliant principles. Today scientists have in their disposal multiple in vivo and in vitro display techniques allowing sophisticated studies and manipulations of a broad range of protein–ligand interactions
eBook - PDFViral Diagnostics
Advances and Applications
- Robert S. Marks, Leslie Lobel, Amadou Sall, Robert S. Marks, Leslie Lobel, Amadou Sall(Authors)
- 2014(Publication Date)
- Jenny Stanford Publishing(Publisher)
300 Phage Display for Viral Diagnostics a potential bioterrorism or biowarfare event. 2 One of the newer techniques being developed for construction of bioreceptors for pathogen detection involves Phage Display technology. “Phage Display,” first introduced by George Smith in 1985, 3 is a powerful technique that allows expression and presentation of peptides or proteins on the phage surface. According to this method, a coding domain of interest is fused to that of a bacteriophage coat protein, resulting in phage particles that display the encoded protein as a chimeric protein. 4, 5 This procedure can also be performed with an ensemble of coding domains, resulting in a phage library that can contain potentially billions of phage variants (up to 10 10 ). 5 In general, the DNA that encodes the displayed protein is encapsulated within the same virion, therefore providing a direct link between phenotype and genotype. 4, 6 This enables rapid amplification and characterization of the desired clone through DNA sequence analysis of the insert. Phage Display has many advantages over other meth-ods for recombinant peptide and protein expression, including ease of manipulation, low production costs, reproducibility, production on a large scale with proper protein folding, and ability to analyze a collection of recombinant molecules to identify those with the highest affinity. 1, 5, 7 Thus Phage Display peptides can be a good alternative to the common method for chemical synthesis of short peptides, the solid-phase peptide synthesis (SPPS), in the case where the presence of the phage itself does not interfere with the system (see Table 14.1). 8 For example, on a 96-well enzyme-linked immunosorbent assay (ELISA) microplate, Phage Display allows a higher number of particles to be coated and also provides better accessibility to the peptide. 9, 10 Several studies have demonstrated the potential of using Phage Display immunogenic peptides or proteins as bases for vaccination.
eBook - ePub- Sheldon J. Park, Jennifer R. Cochran, Sheldon J. Park, Jennifer R. Cochran(Authors)
- 2009(Publication Date)
- CRC Press(Publisher)
1 Phage Display Systems for Protein EngineeringAndreas Ernst and Sachdev S. SidhuCONTENTS- The Phage Display Concept
- Phage Structure and Assembly
- Vectors and Platforms
- The Selection Process
- Evolution of Binding Agents
- Improvement of Protein Stability and Folding
- Identifying Natural Protein–protein Interactions
- Specificity Profiling of Peptide-Binding Modules .
- Mapping Binding Energetics
- Future Perspectives
- References
Information in biological systems is contained and passed on through genes, while the traits of a biological system are given by the functions of encoded proteins and other macromolecules. In molecular biology, the focus of research is the function of biological macromolecules, especially proteins, which are studied in an isolated setting, allowing us to develop our understanding of biological systems. However, these studies are usually limited to a small set of states that can be established in the experiment. With Phage Display, we can study proteins and protein–protein interactions on a combinatorial scale. This means that it is possible to probe billions of different protein variants simultaneously. Phage Display can be applied to the generation of affinity reagents, which are invaluable tools for diagnostics and therapeutic development. The technology can also be used for improving protein stability and for identifying and mapping natural protein–protein interactions in detail.THE Phage Display CONCEPT
Phage Display technology provides an in vitro version of classical Darwinian evolution by establishing a physical linkage between a polypeptide and the encoding genetic information. By mutating DNA encoding for a displayed polypeptide, it is possible to generate a multitude of related variants. This library of variants can be displayed on the surfaces of phage particles, while the DNA encoding for each variant is encased in the phage capsid (Figure 1.1
eBook - PDF- Sheldon J. Park, Jennifer R. Cochran, Sheldon J. Park, Jennifer R. Cochran(Authors)
- 2009(Publication Date)
- CRC Press(Publisher)
1 1 Phage Display Systems for Protein Engineering Andreas Ernst and Sachdev S. Sidhu Information in biological systems is contained and passed on through genes, while the traits of a biological system are given by the functions of encoded proteins and other macromolecules. In molecular biology, the focus of research is the function of biological macromolecules, especially proteins, which are studied in an isolated setting, allowing us to develop our understanding of biological systems. However, these studies are usually limited to a small set of states that can be established in the experiment. With Phage Display, we can study proteins and protein–protein interac-tions on a combinatorial scale. This means that it is possible to probe billions of different protein variants simultaneously. Phage Display can be applied to the genera-tion of affinity reagents, which are invaluable tools for diagnostics and therapeutic development. The technology can also be used for improving protein stability and for identifying and mapping natural protein–protein interactions in detail. THE Phage Display CONCEPT Phage Display technology provides an in vitro version of classical Darwinian evo-lution by establishing a physical linkage between a polypeptide and the encoding genetic information. By mutating DNA encoding for a displayed polypeptide, it is possible to generate a multitude of related variants. This library of variants can be displayed on the surfaces of phage particles, while the DNA encoding for each CONTENTS The Phage Display Concept ....................................................................................... 1 Phage Structure and Assembly ................................................................................... 2 Vectors and Platforms ................................................................................................ 4 The Selection Process ................................................................................................
eBook - ePubPhage Display of Peptides and Proteins
A Laboratory Manual
- Brian K. Kay, Jill Winter, John McCafferty(Authors)
- 1996(Publication Date)
- Academic Press(Publisher)
CHAPTER 2Principles and Applications of Phage Display
Brian K. Kay and Ronald H. HoessINTRODUCTION
The display of peptides and proteins on the surface of bacteriophage represents a powerful new methodology for carrying out molecular evolution in the laboratory. The ability to construct libraries of enormous molecular diversity and to select for molecules with predetermined properties has made this technology applicable to a wide range of problems. The origins of Phage Display date to the mid-1980s when George Smith, on sabbatical in Bob Webster’s laboratory at Duke University, first expressed a foreign segment of a protein on the surface of bacteriophage M13 virus particles. As a test case he fused a portion of the gene encoding the Eco RI endonuclease to the minor capsid protein pIII (Smith, 1985 ). Using a polyclonal antibody specific for the endonuclease, Smith demonstrated that phage containing the Eco RI–gIII fusion could be enriched more than 1000-fold from a mixture containing wild-type (nonbinding) phage with an immobilized polyclonal antibody. From these first experiments emerged two important concepts. First, using recombinant DNA technology, it should be possible to build large libraries (i.e., 108 ) wherein each Phage Displays a unique random peptide. Second, the methodology provides a direct physical link between phenotype and genotype. That is, every displayed molecule has an addressable tag via the DNA encoding that molecule. Because of the ease and rapidity of DNA sequence analysis, selected molecules can be identified quickly. It is interesting to note that the idea of addressable tags is also now being adopted in some combinatorial chemical libraries (Needels et al ., 1993;Ohlmeyer et al ., 1993). Within a few years of George Smith’s experiments the first phage-displayed random peptide libraries were assembled (Cwirla et al ., 1990;Devlin et al ., 1990; Scott and Smith, 1990 ), accompanied by reports that properly folded and functional proteins could also be displayed on the surface of M13 (Bass et al ., 1990;McCafferty et al
- Ronald J. Kendall, Steven M. Presley, Galen P. Austin, Philip N. Smith(Authors)
- 2008(Publication Date)
- CRC Press(Publisher)
One of the more powerful technologies in the field of combina-torial chemistry is Phage Display. 7.3.1 P HAGE D ISPLAY M ETHODOLOGY In 1985, George Smith demonstrated that foreign proteins could be displayed on the surface of filamentous bacteriophage, M13, as a genetic fusion with the gene encod-ing for the capsid protein pIII (Smith 1985) (Figure 7.1). Because the displayed pep-tide is encoded in the viral genome, it was recognized that display technology could provide a powerful tool for selecting and evolving peptide ligands from large combi-natorial peptide libraries (Cortese et al. 1996; McLafferty et al. 1993; Ladner 1995; 182 Advances in Biological and Chemical Terrorism Countermeasures Hosse et al. 2006; Ja et al. 2005). Historically, peptides were displayed as N -terminal fusion peptides with either capsid protein pIII or pVIII (Figure 7.1). However, more recently, employing phagemids (i.e., plasmids carrying phage genes, peptides, and polypeptides) have been displayed from each of the five M13 coat proteins, but not all five on the same phage, suggesting greater flexibility in genetically modifying phage particles and the development of display phage with binding affinities for more than a single target (Russel et al. 2004). The methods of Phage Display technology are based on the general scheme of making a large peptide display library (often involving a repertoire of 10 9 or greater) and putting it though repeated iterations of selection and amplification to select for those peptide ligands that have high affinity for the selected target (Figure 7.2). In selection, those ligands with desired properties (e.g., high affinity) are preferentially separated from the remainder of the library. In amplification, the relatively few selected ligands are copied to form a new generation (i.e., evolution).
eBook - PDFBacteriophages
Perspectives and Future
- Renos Savva(Author)
- 2020(Publication Date)
- IntechOpen(Publisher)
109 Chapter 7 Targeting Peptides Derived from Phage Display for Clinical Imaging Supang Khondee and Wibool Piyawattanametha Abstract Phage Display is a high-throughput technology used to identify peptides or proteins with high and specific binding affinities to a target, which is usually a protein biomarker or therapeutic receptor. In general, this technique allows peptides with a particular sequence to be presented on a phage particle. Peptides derived from Phage Display play an important role in drug discovery, drug delivery, cancer imaging, and treatment. Phage peptides themselves can act as sole therapeutics, for example, drugs, gene therapeutic, and immunotherapeutic agents that are com-prehensively described elsewhere. In this chapter, we discuss phage selection and screening procedures in detail including some modifications to reduce nonspecific binding. In addition, the rationale for discovery and utilization of phage peptides as molecular imaging probes is focused upon. Molecular imaging is a new paradigm that uses advanced imaging instruments integrated with specific molecular imaging probes. Applications include monitoring of metabolic and molecular functions, therapeutic response, and drug efficacy, as well as early cancer detection, personal-ized medicine, and image-guided therapy. Keywords: peptides, membrane receptors, imaging, Phage Display, endoscopy 1. Introduction One of the most important practices in modern era clinical imaging is imaging at the molecular level which can help characterize and measure in vivo biologi-cal processes at the cellular level [1]. Thus, the technique provides unambiguous and high-resolution real-time information for disease diagnoses and therapies.
eBook - ePub- Stefan Dübel, Janice M. Reichert, Stefan D¿bel, Janice M. Reichert, Stefan Dübel, Stefan D¿bel, Janice M. Reichert(Authors)
- 2014(Publication Date)
- Wiley-Blackwell(Publisher)
For this purpose, a panel of technologies were developed such as bacterial surface display [21–23], yeast surface display [24–26], ribosomal display [27–30], or puromycin display [31] (Table 3.1). Despite these manifold technologies, Phage Display became the most widely used selection method. Table 3.1 Comparison of recombinant antibody selection systems Selection system Advantages Disadvantages Transgenic mice Hybridoma technology Somatic hypermutation Immunization required, not freely available Cellular display Bacteria N- and C-terminal and sandwich fusion Not matured, requires individual sorting Yeast Display of larger proteins, N- and C-terminal and sandwich fusion Requires individual sorting Intracelluar display Yeast two hybrid Screening library versus library possible Cytoplasm not optimal for antibody folding Molecular display Puromycin/ribosomal Largest achievable library size in vitro Finicky method Phage Display Filamentous Genomic Robust, multivalent display Prone to mutation, phage production and propagation are coupled, only C-terminal fusion Phagemid Robust, monovalent, and multivalent display by choice of helperphage Only C-terminal fusion T7 Well suited for peptide display No display of antibody fragments, lytic phage Arrays Gridded. clones Robust, simple Small library sizes Modified from Ref. [32]. 3.2 Phage Display An alternative for the generation of human antibodies is antibody Phage Display, which is completely independent of any immune system by utilizing an in vitro selection process. Display systems employing insertion of antibody genes into the phage genome have been developed for phage T7 [33] and phage Lambda [34–36]. However, these systems were not really suitable for antibody generation. The most commonly used display method was developed by George P. Smith [37] on filamentous phage (f1, fd, M13)
- Jeanette M. van Emon(Author)
- 2016(Publication Date)
- CRC Press(Publisher)
Employing biotin-labeled ligands is another selection strategy. Ligand-binding phages can be separated via the biotin moiety using streptavidin-coated magnetic beads [75]. The selectively infective phage (SIP) strategy is a further selection method [76,77]. The receptor protein is fused to C-terminal domains of the pIII coat protein. The recombinant phages are lacking any wild type pIII with the N-terminal N1 domain that is necessary for infection of E. coli . Infectivity is exclusively restored upon the specific interaction of the displayed receptor protein and the ligands that are present in the selection vessel as ligand-N1 fusion construct. Therefore, affinity selection is combined with the capability for reinfection. 2.4.2 C ELL S URFACE D ISPLAY In cell surface display, the protein library is fused to cellular membrane proteins of bacteria, yeast, or mammalian cells [78]. The membrane protein, usually a lipoprotein, is anchored in the cell membrane and presents the desired protein on the cell surface (cf. Figure 2.6). For instance, a common anchor protein utilized in yeast display is the cell surface receptor a-agglutinin [68]. Protein selection is performed by fluorescence-activated cell sorting (FACS). The ligand is labeled for this purpose with fluorescent markers [79]. State-of-the-art flow cytometers can analyze and sort 50,000 cells per second, providing a rapid and high performance sampling of receptor libraries [78]. Directed Evolution of Ligand-Binding Proteins 55 2.4.3 R IBOSOME D ISPLAY Ribosome display is an in vitro display system [69,80,81]. In vitro display libraries comprise up to 10 14 different proteins. In contrast to phage or surface display on bacteria and yeast, the in vitro approach does not depend on a transformation step for creating a selectable library of protein variants. Therefore, the size is not limited by the transformation efficiency of DNA into a host organism.
eBook - PDF- Thomas Böldicke(Author)
- 2018(Publication Date)
- IntechOpen(Publisher)
British Journal of Cancer. 2000; 83 (2):252-260 [92] Zhao A, Tohidkia MR, Siegel DL, Coukos G, Omidi Y. Phage antibody display librar-ies: A powerful antibody discovery platform for immunotherapy. Critical Reviews in Biotechnology. 2016; 36 (2):276-289 Display Technologies for the Selection of Monoclonal Antibodies for Clinical Use http://dx.doi.org/10.5772/intechopen.70930 69 [93] Boder ET, Wittrup KD. Yeast surface display for screening combinatorial polypeptide libraries. Nature Biotechnology. 1997; 15 (6):553-557 [94] Feldhaus MJ, Siegel RW. Yeast display of antibody fragments: A discovery and charac-terization platform. Journal of Immunological Methods. 2004; 290 (1-2):69-80 [95] Gera N, Hussain M, Rao BM. Protein selection using yeast surface display. Methods. 2013; 60 (1):15-26 [96] Sheehan J, Marasco WA. Phage and yeast display. Microbiology Spectrum. 2015; 3 (1):17 AID-0028-2014 [97] Shusta EV, Kieke MC, Parke E, Kranz DM, Wittrup KD. Yeast polypeptide fusion sur -face display levels predict thermal stability and soluble secretion efficiency. Journal of Molecular Biology. 1999; 292 (5):949-956 [98] Shusta EV, Holler PD, Kieke MC, Kranz DM, Wittrup KD. Directed evolution of a stable scaffold for T-cell receptor engineering. Nature Biotechnology. 2000; 18 (7):754-759 [99] Orr BA, Carr LM, Wittrup KD, Roy EJ, Kranz DM. Rapid method for measuring ScFv thermal stability by yeast surface display. Biotechnology Progress. 2003; 19 (2):631-638 [100] Boder ET, Midelfort KS, Wittrup KD. Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Proceedings of the National Academy of Sciences of the United States of America. 2000; 97 (20):10701-10705 [101] Feldhaus MJ, Siegel RW, Opresko LK, Coleman JR, Feldhaus JM, Yeung YA, et al. Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library.
eBook - ePub- Stefan Lutz, Uwe Theo Bornscheuer(Authors)
- 2012(Publication Date)
- Wiley-VCH(Publisher)
Phages are ‘sticky’ towards themselves and towards solid supports and, when concentrated, can form soluble aggregates that will dissociate relatively slowly. Therefore it is recommended that all phage solutions be vortexed thoroughly before infection. As the phage will also stick to micropipette tips, the tips should be changed when performing serial dilutions. Finally, when the phages are highly diluted, a time-dependent loss of infection may result from their binding to the vessel walls. Hence, it is recommended either that silanized microtubes are used, that 1% BSA is added to the solution, or that highly diluted solutions should not be kept for long periods of time.24.3.12.2 Displayed Protein is Degrading with Time
This effect may be due to the presence of proteases or to an inherently low protein stability. A ‘cocktail’ of protease inhibitors can be added (Complete tabs, Roche), and freshly prepared phage solutions should always be added when performing selection from libraries.24.3.12.3 Phages are not Genetically Stable
This may be due to a low toxicity of the fusion protein or to recombination with homologous E. coli genes. The solution is to use a recA strain such as JM109 to reduce recombination. To avoid problems of toxicity, a phagemid vector such as pHDi.Ex [92] should be used, as this allows control of the fusion protein expression. With this vector, a strong repression will be obtained by adding 1% glucose (catabolic repression).24.3.12.4 The Ratio ‘Out/In’ is not Increasing with the Selection Rounds
This may mean that no clones are being selected. For some strategies where low-affinity capture may be necessary for selection, the level of specifically captured phages may always be below the background level. It is therefore worth analyzing the selected phages as an effective enrichment may have occurred.24.4 Conclusions and Future Challenges
In less than 25 years, Phage Display has become a robust and extremely powerful technology for creating artificial protein binders that could find applications both in fundamental research and industrial processes. The first therapeutic antibodies evolved by Phage Display are now available commercially, more are in clinical trials and, most certainly, many more will follow. There is no doubt that the use of antibodies will continue to increase in the near future, although alternative scaffolds are emerging with attractive advantages such as small size, a high level of recombinant expression or intracellular applicability. Now, with a good scaffold and a good library, the selection of strong binders is not exceptional, perhaps even easy, and alternatives to antibodies are changing their status from small outsiders to serious competitors. Even more promising, the engineering of bifunctional proteins that combine a binding site with a fluorescent property, an enzymatic active site or another binding site is probably the next major challenge. These proteins would be capable of both recognizing a target and transmitting a signal, and should find useful applications in diagnostics or immunotherapies.
eBook - PDF- William Wu, Helen H. Zhang, Michael J. Welsh, Peter B. Kaufman(Authors)
- 2016(Publication Date)
- CRC Press(Publisher)
All three forms, Fv, scFv, and FAb, have been expressed on the surface of phage. 5–6 A number of functional antibodies have been successfully expressed on the surface of phage, including alkaline phosphatase, protein A, CD4, and growth hormone. 4–11 There are conservative sequences within the variable regions of antibody genes among species. Alignment and analysis of variable domains can lead to the deter-mination of species-specific consensus sequences, which will be used for design of polymerase chain reaction (PCR) primers. Hence, the whole repertoires of antibody genes can be prepared by PCR. In this way, recombinant phage antibody technology has the power and versatility to mimic the features of immune diversity and mono-clonal selection. Phage Display of recombinant antibodies takes advantages of the unique features of the M13 phage life cycle and the expression of a couple of phage proteins on the surface of the M13 phage. 2–4 The M13 phage is filamentous and approximately 895 nm long and 9 nm in diameter. Its genome is single-stranded DNA that contains 6.4 kb encoding 10 different proteins. The genome encodes a coat protein composed of approximately 2700 copies of the gene 8 protein (g8p). I, the M13 phage expresses three to five copies of the gene 3 adsorption protein (g3p) on its tip. 2–3 Phage Display technology utilizes the features of g3p and engineered recombinant gene III, gener-ating fusion g3p and expression of recombinant proteins or antibodies on the surface of the M13 phage. When these antibodies bind to the antigen, they bind the phage to the antigen as well, indicating that antigen-reactive antibodies are expressed on the tips of the M13 phage. The phage particles can be detected by an enzyme-labeled antibody against the g8 coat proteins (g8p). M13 phage does not produce lytic infection in Escherichia coli as seen in most of other bacteriophage life cycle.
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