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.
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.tubs.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/i
mages/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/i
mages/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/p
artnerships/biology/200710/images/ker
r_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/im
age/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
