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Phage Display

Phage Display. Anna Snapp. What is phage display?. An in vitro selection technique using a peptide or protein genetically fused to the coat protein of a bacteriophage. http://www.bio.davidson.edu/people/dawessner/micro/images/bacteriophage.gif. A Bacterio What?.

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Phage Display

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  1. Phage Display Anna Snapp

  2. What is phage display? An in vitro selection technique using a peptide or protein genetically fused to the coat protein of a bacteriophage. http://www.bio.davidson.edu/people/dawessner/micro/images/bacteriophage.gif

  3. A Bacterio What? • Bacteriophages are viruses that infect bacterial cells. • Infected cells are used as hosts to replicate the virus. • E. coli phages used due to ease of culture and quick regeneration. http://library.thinkquest.org/C0123260/basic%20knowledge/images/basic%20knowledge/DNA/structure

  4. Life cycle of phage http://rzv054.rz.tu-bs.de/Biotech/SD/m13LiveCycle.jpg

  5. Phage Display Libraries • E. coli can be infected to multiply the number of bacteriophages. • Can quickly create large libraries of phage clones displaying different peptides. http://ifa.hawaii.edu/~jrich/oldstuff/tens/bacteriophage2.jpg

  6. Steps Involved In Using Phage Display • Creation of vector • Binding/Selection • Wash • Elution • Amplification

  7. Creation of Viral Vectors • Recombinant DNA technology to incorporate foreign cDNA of interest into viral DNA. • Spliced into gene for a coat protein so the protein will be displayed on outside of phage particles

  8. Incorporation of the VH/K gene protein into the phage coat proteins. Gene VIII is a phage coat protein gene. http://www.freepatentsonline.com/6586236-0-large.jpg

  9. Advantages of Coat Protein Display for Selection • Allows for a direct link between the DNA sequence and protein. • Exposed to solvent so the protein can retain its affinities and functions. http://dennehylab.bio.qc.cuny.edu/images/14_color_phage.jpg

  10. Binding and Selection aka Biopanning • Can apply standard affinity techniques to capture phage by taking advantage of displayed proteins. • Pass solutions of amplified phages over solid support with antigens or receptors bound to it. • Phages with affinity to support bind.

  11. Next Step: Wash • Unbound phages are washed away leaving only those showing affinity for the receptors. http://www.luainnovations.com/technologies/images/phages.jpg

  12. Elution! • Bound phages can be eluted by disrupting the protein bonding interactions. • Acidic buffers, Alkaline buffers, Urea, addition of soluble ligand for receptor. • Can also add host cells to infect

  13. Amplification • Eluted phages showing specificity are used to infect new host cells for amplification. • Cycle repeated 2-3 times for stepwise selection of best binding sequence. http://www.washington.edu/alumni/partnerships/biology/200710/images/kerr_ecoli2.jpg

  14. Analyze • Final phages can be propagated then characterized with DNA sequencing. • Common motifs involved with binding may emerge for further study.

  15. Phage Display cycle Hoogenboom et al.

  16. Applications • Epitope mapping and mimicking • Identification of new receptors & ligands • Drug discovery • Epitope discovery – new vaccines • Creation of antibody libraries • Organ targeting

  17. Epitope mapping and mimicking • Use random libraries to determine if it is continuous • Compare phage sequence motif to amino acid sequence of natural ligands • Map critical binding sites of epitope/ligands

  18. Identification of new receptor ligands • Can identify new receptors that bind the same ligand. • Can use to study signal proteins and pathways – link • Match receptor with unknown ligand http://www.apsnet.org/online/feature/phages/image/phage4sm.jpg

  19. Drug Discovery • Test receptors as targets of drugs • Peptides can act as antagonists, agonists, or modulators • Large scale search but might not have good pharmacological properties

  20. Epitope Discovery • Use antibodies as a receptor to select peptide that is an antigen mimic. • Use mimic to immunize and elicit antibody increase (immunogenic mimic) • Can bypass animal immunization by mimicking immune selection.

  21. Organ Targeting • Help ID endothelial cell selective markers that target cells to help get drugs to selected tissue. • Inject phage into mouse then extract phages from different organs. • Identify common motifs possibly involved with localization.

  22. Phage Display Overview:Propagate, Bind, Wash, Elute, Repeat http://www.bioscience.org/2008/v13/af/2749/fig2.jpg

  23. Advantages of Phage Display • Easy to screen large # of clones >109 • Easy to amplify selected phages in E. coli • Selection process easy and already in use in various forms. • Can create Phage library variation by inducing mutations, using error prone PCR, etc.

  24. Disadvantages • Might not have long enough peptide insert so critical folding can be disrupted. • Could lose phage variations if first bind/wash step too stringent. • Affinities or binding that results during selection might not work in vivo.

  25. References • George P. Smith, Valery A. Petrenko. Phage Display. Chemical Reviews. 1997. 97(2) pp. 391-410 • Tim Clackson, Hennie R. Hoogenboom, Andrew D. Griffiths, Greg Winter. Making antibody fragments using phage display libraries. 1991 Nature vol 352 • RenataPasqualini, ErkkiRuoslahti. Organ Targeting in vivo using Phage display peptide libraries. 1996 Nature vol 380. • Hennie R. Hoogenboom, Adriaan R. deBruine, Simon E. Hufton, Rene M. Hoet, Jan-Willem Arends, Rob C. Roovers. Antibody phage display technology and its applications. 1998 Immunotechnology vol4 issue1 pg.1-20 • New England Biolabs FAQ’s Phage Display Peptide Libraries. 2007 http://www.neb.com/nebecomm/tech_reference/protein_tools/phdfaq.asp

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