Phage
Display
Phage Display as a Tool for Protease Ligand Discovery Pp-1-12
Andrew E.
Nixon
Direct Selection of cDNAs from Filamentous Phage Surface Display Libraries: Potential and Limitations Pp-13-21
Claudio
Rhyner, Rimantas Kodzius and Reto Crameri
Bacteriophage Lambda as a Vehicle for Peptide and Protein Display Pp-23-28
Ronald H.
Hoess
Application of Phage Display Technology to Cancer Research Pp-29-43
Yum L.
Yip and Robyn L. Ward
Evolving Phage Vectors for Cell Targeted Gene Delivery Pp-45-57
David
Larocca, Michael A. Burg, Kristen Jensen-Pergakes, Edward Prenn Ravey, Ana
Maria Gonzalez, and Andrew Baird
[Back to
top] Phage Display as a Tool for Protease Ligand
Discovery
Andrew E.
Nixon
Proteolytic
enzymes have been implicated as the pathological agent in a number of disease
states. For this reason proteases are attractive therapeutic targets. Phage
display of peptide libraries can be used to identify peptides that may be used
either directly as inhibitors or serve as leads in the generation of prodrugs
and peptidomimetics.
[Back to
top] Direct Selection of cDNAs from Filamentous Phage
Surface Display Libraries: Potential and Limitations
Claudio
Rhyner, Rimantas Kodzius and Reto Crameri
Over
the past decade, powerful technologies devoted to survey large molecular
libraries for the presence of specific clones using the discriminative power of
affinity selection have been developed. Phage surface display technology is the
most established of these methods and has revolutionised our ability to select
agonistic and antagonistic peptides, antibodies with desired specificity and
other drug targets. Thereby ligands are expressed as fusions to phage coat
proteins and their respective genes are packaged within the phage. The basic
concept of linking the phenotype, expressed as gene product displayed on the
phage coat, to its genetic information integrated into the phage genome,
creates fusion proteins covalently associated with the infectious particle
itself. Binding of the phage to the target molecule offers a selective system
by which rare phage carrying the desired gene product can be selected from
large phage populations carrying inappropriate sequences. Phage selected in
this fashion can be used for subsequent rounds of selection because they are
able to self-replicate in suitable host cells, yielding target-specific phage
populations after several consecutive rounds of affinity selection. Over 1500
publications describe the use of phage display technology highlighting its
performance. Phage display possesses certain limitations, including its use for
selection of genes from cDNA libraries that has lagged behind, despite the many
accomplishments of this technology. Here we discuss recent progress in construction
and screening of cDNA libraries displayed on phage surface and emerging
concepts allowing fast identification of virtually all different clones present
in enriched libraries.
[Back to
top] Bacteriophage Lambda as a Vehicle for Peptide and
Protein Display
Ronald H.
Hoess
Bacteriophage
lambda has emerged as an alternative vehicle for the surface display of
peptides and proteins to the commonly used filamentous phage. There are a
number of unique features that make lambda an attractive display vehicle
including the ability to display multimeric proteins, no requirement for
secretion of the displayed fusion protein and the means to vary the valency of
the displayed fusion protein. With its increasing use for cDNA encoded display,
the lambda display systems will be an important tool for functional genomics.
[Back to
top] Application of Phage Display Technology to
Cancer Research
Yum L.
Yip and Robyn L. Ward
Despite
years of international effort, cancer remains a major cause of death in
developed countries, claiming more than 500 000 lives per year in the United
States alone. Recombinant DNA technology and high throughput screening methods
have recently increased the pace of cancer research. In this review, we will
examine the impact and contribution of phage display technology to this area of
research. As a biological combinatorial system, the strength of phage display
lies in its flexibility and its ability to efficiently study protein-protein
interactions. The technology has also facilitated the discovery of molecules
that have potential roles in the diagnosis and treatment of cancer.
[Back to
top] Evolving Phage Vectors for
Cell Targeted Gene Delivery
David
Larocca, Michael A. Burg, Kristen Jensen-Pergakes, Edward Prenn Ravey, Ana
Maria Gonzalez, and Andrew Baird
We
adapted filamentous phage vectors for targeted gene delivery to mammalian cells
by inserting a mammalian reporter gene expression cassette (GFP) into the
vector backbone and fusing the pIII coat protein to a cell targeting ligand
(i.e. FGF2, EGF). Like transfection with animal viral vectors, targeted phage
gene delivery is concentration, time, and ligand dependent. Importantly,
targeted phage particles are specific for the appropriate target cell surface
receptor. Phage have distinct advantages over existing gene therapy vectors
because they are simple, economical to produce at high titer, have no intrinsic
tropism for mammalian cells, and are relatively simple to genetically modify
and evolve. Initially transduction by targeted phage particles was low
resulting in foreign gene expression in 1-2% of transfected cells. We increased
transduction efficiency by modifying both the transfection protocol and vector
design. For example, we stabilized the display of the targeting ligand to
create multivalent phagemid-based vectors with transduction efficiencies of up
to 45% in certain cell lines when combined with genotoxic treatment. Taken
together, these studies establish that the efficiency of phage-mediated gene
transfer can be significantly improved through genetic modification. We are
currently evolving phage vectors