phage display technology its importance

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PRESENTATION ON
PHAGE DISPLAY TECHNOLOGY
Prepared By:
Balaji Rathod
PGS16AGR7165

2.  Phage display technology has emerged in 1985 by
George Smith.
 Phage display is a laboratory technique for the study
of protein–protein, protein–peptide, and protein–
DNA interactions that uses bacteriophages to connect
proteins with the genetic
information that encodes them.
 In this technique, a gene encoding a protein of interest
is inserted into a phage coat protein gene, causing the
phage to "display" the protein on its outside while
containing the gene for the protein on its inside,
resulting in a connection
between genotype and phenotype. 2

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 In this way, large libraries of proteins can be screened
and amplified in a process called in vitro selection, which
is analogous to natural selection.
 These displaying phages can then be screened against
other proteins, peptides or DNA sequences, in order to
detect interaction between the displayed protein and those
other molecules.

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 The most common bacteriophages used in phage display
are filamentous phages viz., fd, f1, M13

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12.  They are more easily cloned and screening
 They do not require animals / cell culture
 They offer stable genetic sources
 They can be genetically manipulated
 purification is easy
 Cheap, simple, rapid and requires no special equipment
 They are useful as immunological agents
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13.  Long, thin, and flexible particles of
930
nm in length and 6.5 nm in diameter
 Consists of circular single-strand DNA
of 6407 nucleotides long
 With 11 genes
 The major coat protein is pVIII
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14.  The minor coat proteins pIII and pVI
are located at one end of the phage;
 pVII and pIX are located at the other
end of the phage
 It infects gram-negative bacteria
 Its non-lysogenic characteristic to
permits the extrusion of recombinant
phage into the culture supernatant
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 Antibody fragments used in phage display

16.  The DNA encoding the protein or peptide of interest
is ligated into the pIII or pVIII gene, encoding either the
minor or major coat protein, respectively.
 The phage gene and insert DNA hybrid is then inserted (a
process known as "transduction") into Escherichia coli (E.
coli) bacterial cells.
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17.  By immobilizing a relevant DNA or protein target(s) to the
surface of a micro titer plate well, a phage that displays a
protein that binds to one of those targets on its surface will
remain while others are removed by washing.
 Those that remain can be eluted, used to produce more
phage (by bacterial infection with helper phage) and so
produce a phage mixture that is enriched with relevant (i.e.
binding) phage.
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18.  The repeated cycling of these steps is referred to
as 'panning', in reference to the enrichment of a sample of
gold by removing undesirable materials.
 Phage eluted in the final step can be used to infect a
suitable bacterial host, from which the phagemids can be
collected and the relevant DNA sequence excised and
sequenced to identify the relevant, interacting proteins or
protein fragments.
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Phage display cycle

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Displaying Targets on
Filamentous Phage
Panning (Selection) of
The Phage Library
Expression the Protein Fragment
or Isolation of Affinity-Purified
Phage Clones
Library clones
Expression
Isolation

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selection and screening

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 The sources of genetic material

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 The proteins that are created and isolated by phage display
process have a specific interaction with a known disease
target,making this a rapid, effective and focused drug
discovery method. The proteins are candidates for
effective drug therapies.

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 . The natural protein binding partners of a given target may be identified by isolating
phage displayed peptides that bind to the target and comparing them to a database of
native sequences.

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 Directed evolution Phage display is a powerful tool for molecular evolution.

42.  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.

43.  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.

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