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

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

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

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

Creation of vector Binding/Selection Wash Elution Amplification

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

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

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

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.

Unbound phages are washed away leaving only those showing affinity for the receptors.

http://www.luainnovations.com/technologies/images/phages.jpg

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

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

Final phages can be propagated then characterized with DNA sequencing.

Common motifs involved with binding may emerge for further study.

Hoogenboom et al.

Epitope mapping and mimicking Identification of new receptors &

ligands Drug discovery Epitope discovery – new vaccines Creation of antibody libraries Organ targeting

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

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

Test receptors as targets of drugs Peptides can act as antagonists,

agonists, or modulators Large scale search but might not have

good pharmacological properties

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.

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.

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

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.

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.

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