2. 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.  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. http://rzv054.rz.tu- bs.de/Biotech/SD/m13LiveCycle.jpg

5.  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.  Creation of vector  Binding/Selection  Wash  Elution  Amplification

7.  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.  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/i mages/14_color_phage.jpg

10.  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.  Unbound phages are washed away leaving only those showing affinity for the receptors. http://www.luainnovations.com/technologies/i mages/phages.jpg

12.  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.  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/p artnerships/biology/200710/images/ker r_ecoli2.jpg

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

15. Hoogenboom et al.

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

17.  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.  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/im age/phage4sm.jpg

19.  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.  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.  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. http://www.bioscience.org/2008/v13/af/2749/fig2.jpg

23.  Easy to screen large # of clones >10 9  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.  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.  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  Renata Pasqualini, Erkki Ruoslahti. 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 http://www.neb.com/nebecomm/tech_reference/protein _tools/phdfaq.asp