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

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

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

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 genein the middle section of the gene III.


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.

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 libraryphagemid construction

transformation E. coli pool containing different phages

helper phage transduction

phage poolscreening transduction

Cells with desired DNA


leucine zipper reinforced with disulphide bonds

leucine zipper, aka leucine scissors

• Each half of a leucine zipper co

nsists of a short alpha-helix wit

h a leucine residue at every sev

enth position. The standard 3.

6-residues-per-turn alpha-helix

structure changes slightly to be

come a 3.5-residues-per-turn al

pha-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).


Targeted Molecular Imaging

Peptide-Based Probes for Targeted Molecular ImagingSeulki Lee, Jin Xie, and Xiaoyuan Chen ,Biochemistry 2010, 49, 1364–1376

• Although some success has been achieved, the use of these probes has bee

n largely unsuccessful mainly because of their low specificity (small molec

ules) 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 contr

ast in vivo. The ideal imaging compound would exhibit high binding affini

ty for the target, specific uptake and retention in the target, rapid clearance

from nontarget tissue, adequate capillary permeability, and high stability a

nd integrity in vivo and would be easy to prepare and safe for use.

Peptide-Based Probes for Targeted Molecular ImagingSeulki Lee, Jin Xie, and Xiaoyuan Chen ,Biochemistry 2010, 49, 1364–1376

Combinatorial peptide chemistry and phage display te

chnology, a molecular genetics approach to ligand dis

covery, have profoundly impacted the pool of availab

le bioactive synthetic peptides and peptide hormones.

Combinatorial peptide chemistry

Combinatorial libraries of peptide dendrimers: design, synthesis, on-bead high-throughput screening, bead decoding and characterizationNoélie Maillard, Anthony Clouet, Tamis Darbre & Jean-Louis ReymondNature Protocols 4, 132 - 142 (2009) Published online: 15 January 2009doi:10.1038/nprot.2008.241

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

This method inserts the randomized

oligonucleotide library DNA in-frame

after a.a. 348 of capsid 10B gene.

phage-displayed peptide libraries

Documents

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