Download Our work was originally motivated my collaboration with Drs

Our work was originally motivated my collaboration with Drs
Pasqualini & Arap who have been studying molecular
targets that may be used to direct therapies to specific
tissues. The long term goal of this kind of work is that if
drugs can be targeted to specific tissues in the body, then
dosage can be altered to achieve the desired effect while
minimizing side effects such as toxicity.
The assumption that different tissues can be targeted
presupposes the existence of biochemical differences which
can be exploited. The type of difference that we are looking
for is a differential concentration of protein binding sites:
something that says that if you inject protein A into the body,
the only tissue to which that protein can bind is tissue A.
In vivo phage display is a technology used to reveal organ
specific vascular ligand-receptor systems in animal models,
and recently in patients, and also to validate them as
potential therapy targets.
Phage display is a biological process to individually display
up to millions of peptides and proteins on the surface of a
small bacterial virus -- a phage. The use of phage display in
screening for high-affinity ligands and their receptors has
been crucial in functional genomics and proteomics.
A bacteriophage library can be constructed to provide the
artificial receptors. The library can contain millions of
bacteriophage with randomly displayed peptide sequences
in the phage outer protein coat which act as binding sites for
the agents of interest. This library will be used to 'bio-pan' for
phages that bind to a number of toxins and infectious agents
and can, thus, provide an endless supply of low cost,
reliable, specific, and stable artificial receptors.
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Phage display describes a selection technique in which a library
of variants of a peptide or protein is expressed on the outside of
a phage virion, while the genetic material encoding each variant
resides on the inside (1-3). This creates a physical linkage
between each variant protein sequence and the DNA encoding
it, which allows rapid partitioning based on binding affinity to a
given target molecule (antibodies, enzymes, cell-surface
receptors, etc.) by an in vitro selection process called panning
(4). In its simplest form (Figure 1), panning is carried out by
incubating a library of phage-displayed peptides with a plate (or
bead) coated with the target, washing away the unbound
phage, and eluting the specifically bound phage. The eluted
phage is then amplified and taken through additional
binding/amplification cycles to enrich the pool in favor of binding
sequences. After 3-4 rounds, individual clones are
characterized by DNA sequencing and ELISA.
Figure 1: Panning with a Ph.D. Phage Display Library
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Peptides are composed of amino acids, and the length
of a peptide is defined as the number of amino acids in
the peptide. For example, a tri-peptide has three amino
acids. There are 20 different amino acids, which leads
to 4,200 distinct tri-peptides (not counting mirror
images, e.g., ABC is the same as CBA). For quadripeptides, the number of distinct types is approximately
20 times larger. We are looking for differential binding
profiles by using specially designed phage probes. A
DNA sequence corresponding to a known peptide
sequence is spliced into the phage DNA. When this
spliced sequence is transcribed and translated it
produces a 9-mer polypeptide of the form $CX_7C$
(here, $C$ = cysteine, $X$ = any amino acid) which is
attached to one of the phage's coat proteins.
Conceptually, there are strands extruded from the surface of
the phage which terminate in the given polypeptide
sequence. The two cysteines at the ends tend to bind to
each other, forming a loop. If these loops encounter suitable
binding sites, the phages will attach. The experiment
proceeds as follows: a large number of phages with diverse
9-mers are injected into the subject and allowed to disperse
through the circulatory system. Shortly there after, samples
are taken from target tissues of interest. These samples are
examined for phage; each phage that is found is cultured
and its specific 9-mer sequence determined. Repetitive
motifs concentrated in the phage sampled from a particular
tissue may indicate the higher availability of a specific type of
binding site.
The case study we work with involves 3 stages of mouse
filtering. We begin with an unselected cyclic random peptide
library CX_7C injected into mice. Six organs were targeted
in mice: muscle, bowel uterus, kidney, pancreas, brain. At
each stage, peptide-displaying phage are isolated from
target organs, amplified, and pooled for the next round of
selection. The total number of tri-peptides from a small pool
is obtained by randomly sampling from the phage library
harvested from the mouse organs. the tri-peptide counts
from the phage library are expected to increase across the
multiple stages of the experiment if they are to bind to
specific organs with high affinity. Likewise, the counts for the
tri-peptides that do not bind will stay unchanged or even
decrease, since only a certain number of peptides may bind
to an organ.