Phage display
• Phage display is a term describing display
of foreign (poly)peptides on the surface of
phage particle. This is achieved by splicing
a gene encoding such a peptide into a
gene encoding a capsid structural protein.
• Phage Display was originally invented by
George P. Smith in 1985
filamentous phage: M13, Ffphage
Smith demonstrated that fusions to the capsid protein p3
of the non-lytic filamentous phage f1
were fairly well tolerated by cloning
a fragment of the EcoRI restrictase gene
in the middle section of the gene III.
Progress in phage display: evolution of the technique and its applications
Tomaž Bratkovič
Cellular and Molecular Life Sciences, 2009 10.1007/s00018-009-0192-2
Using recombinant DNA technology,
collections of billions of peptides,
protein variants, gene fragment- or
cDNA-encoded proteins
presented on phage
(so-called phage-displayed libraries)
can be constructed and surveyed
for specific affinity or activity.
Progress in phage display: evolution of the technique and its applications
Tomaž Bratkovič
Cellular and Molecular Life Sciences, 2009 10.1007/s00018-009-0192-2
phagemid
Phagemids combine features of plasmids
(i.e., carry antibiotic resistance and
enable replication of dsDNA)
with features of phage vectors
(i.e., allow for production and
packing of ssDNA into virions).
helper phage
• Phagemids are engineered to express recombinant p3
fusions under controlled conditions, but do not encode
any viral structural or replication proteins.
• Only upon superinfection of a bacterial host by a helper
phage which contributes the missing genes can the
phagemid ssDNA replication and packing take place.
•
Helper phage are defective in their origin of replication
or packaging signal thereby ensuring preferential
packing of phagemids.
cDNA library
transformation E. coli pool containing
phagemid construction
different phages
screening
phage pool
Cells with desired DNA
transduction
helper phage
transduction
leucine zipper reinforced with disulphide bonds
Progress in phage display: evolution of the technique and its applications
Tomaž Bratkovič
Cellular and Molecular Life Sciences, 2009 10.1007/s00018-009-0192-2
leucine zipper, aka leucine scissors
• Each half of a leucine zipper
consists of a short alpha-helix
with a leucine residue at every
seventh position. The standard
3.6-residues-per-turn alphahelix structure changes slightly
to become a 3.5-residues-per-
turn alpha-helix.
• Schematic representation of antibody fragment types
displayed as fusions to p3: a single-chain variable
fragment (scFv); b antigen-binding fragment (Fab)
with light chain-p3 fusion (left) or heavy chain-p3
fusion (right).
Progress in phage display: evolution of the technique and its applications
Tomaž Bratkovič
Cellular and Molecular Life Sciences, 2009 10.1007/s00018-009-0192-2
Targeted Molecular Imaging
Peptide-Based Probes for Targeted Molecular Imaging
Seulki Lee, Jin Xie, and Xiaoyuan Chen ,Biochemistry 2010, 49, 1364–1376
• Although some success has been achieved, the use of these probes has been
largely unsuccessful mainly because of their low specificity (small
molecules) or limited target permeability (antibodies).
• For an imaging probe to be clinically useful, it should provide a sufficient
“target-to-background” ratio to maximize the “signal-to-noise” ratio or
contrast in vivo. The ideal imaging compound would exhibit high binding
affinity for the target, specific uptake and retention in the target, rapid
clearance from nontarget tissue, adequate capillary permeability, and high
stability and integrity in vivo and would be easy to prepare and safe for use.
Peptide-Based Probes for Targeted Molecular Imaging
Seulki Lee, Jin Xie, and Xiaoyuan Chen ,Biochemistry 2010, 49, 1364–1376
Combinatorial peptide chemistry and phage display
technology, a molecular genetics approach to ligand
discovery, have profoundly impacted the pool of
available bioactive synthetic peptides and peptide
hormones.
Combinatorial peptide chemistry
Combinatorial libraries of peptide dendrimers: design, synthesis,
on-bead high-throughput screening, bead decoding and characterization
Noélie Maillard, Anthony Clouet, Tamis Darbre & Jean-Louis Reymond
Nature Protocols 4, 132 - 142 (2009) Published online: 15 January 2009
doi:10.1038/nprot.2008.241
phage-displayed peptide libraries
This method inserts the randomized
oligonucleotide library DNA in-frame
after a.a. 348 of capsid 10B gene.
T7 lytic phage-displayed peptide libraries exhibit less sequence bias than
M13 filamentous phage-displayed peptide libraries
Lauren R. H. Krumpe, Andrew J. Atkinson, GaryW. Smythers, Andrea Kandel
Kathryn M. Schumacher, James B. McMahon, Lee Makowski and Toshiyuki Mori
Proteomics 2006, 6, 4210–4222 DOI 10.1002/pmic.200500606