Mar. 31 Presentation Phage Display

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Transcript Mar. 31 Presentation Phage Display

An in vitro selection technique using a
peptide or protein genetically fused to
the coat protein of a bacteriophage.
Bacteriophages are
viruses that infect
bacterial cells.
 Infected cells are used
as hosts to replicate the
 E. coli phages used due
to ease of culture and
quick regeneration.
E. coli can be infected
to multiply the number
of bacteriophages.
 Can quickly create
large libraries of phage
clones displaying
different peptides.
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
of the VH/K
gene protein
into the
phage coat
Gene VIII is a
phage coat
protein gene.
Allows for a direct link
between the DNA
sequence and protein.
Exposed to solvent so the
protein can retain its
affinities and functions.
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.
Bound phages can be eluted by
disrupting the protein bonding
› 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
 Cycle repeated 2-3 times for stepwise
selection of best binding sequence.
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
 Compare phage sequence motif to
amino acid sequence of natural ligands
 Map critical binding sites of
Can identify new receptors that bind the
same ligand.
 Can use to study signal proteins and
pathways – link
 Match receptor with unknown ligand
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.
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