The document discusses phage display technology. It introduces phage display and describes the biology of filamentous phages like M13 that are commonly used. It explains how phagemids combine features of plasmids and phages to allow expression of recombinant proteins. Different types of phage display systems are described, including types that display antibody fragments. The process of biopanning and screening phage display libraries to isolate antibodies with high affinity for a target is summarized in three steps: production of the library, selection through biopanning, and screening candidates. Applications of phage display technology including production of monoclonal antibodies are also mentioned.

1. • Introduction of Phage Display Technology
• General view to biology of filamentous phages
• Biology of Phagemids
• Types of host & process of infection
• Display of antibody fragments
• Types of phage display systems
• Phage-display cycle(Biopanning & Screening)
• Phage display libraries
• Application of phage display

3.  Filamentous phages are group of non-lytic
phages that
 incorporate round single stranded DNA. The
family of Ff, M13,
 fd, and f1 are vital phages which have utility
in phage display
 which among them M13 phage is the most
generally used.

4.  M13 phage, is a bacteriophage that is (6–7) nm in diameter
and 900 nm long and is a member of F positive family. The
genome of M13 phage is 6407 bp long that encodes three
groups of phage proteins:
 1 –proteins involving in replication (pII, pX and pV),
 2 –morphogenetic (pI and pIV), and
 3 – structural (pVIII, pIII,pVI, pVII and pIX). The coating
proteins of phage categorized into major (pVIII) and minor
(PIII and pVI) proteins.
 Each phage has about 2700 copies of pVIII. The N-terminal
region of pIII plays important role in binding of phage to
the F-pilus of the bacterium [13–15].

12.  Phagemids are Ff-phage-derived vectors, containing the
replication origin of a plasmid. The basic components of a
phagemid mainly include the replication origin of a
plasmid, the selective marker, the intergenic region (IG
region, usually contains the packing sequence and
replication origin of minus and plus strands), a gene of a
phage coat protein, restriction enzyme recognition sites, a
promoter and a DNA segment encoding a signal peptide .
Additionally, a molecular tag can be included to facilitate
screening of phagemid-based library. The functions of
these elements will be discussed in Construction
Strategies and Characteristics of Phagemids in
detail.Phagemids can be converted to filamentous phage
particles with the same morphology as Ff phage22 by co-
infection with the helper phages,23such as
R408,24M13KO725and VCSM13.

15. 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).

16.  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.

17.  The amber codon TAG can be inserted between
foreign genes and coat protein genes .
 host strains 1: in E. coli supE strains such as XL-1 Blue
MRFV, TG1, DH5aFV, ER2738, ER2537 and 16C9, TAG codon is
recognized as glutamic acid codon instead of stop codon. As a
result, the foreign protein is expressed as a fusion protein
presented on the surface of progeny phages.
2 : In the non-suppressor strains such as TOP10F', JS5
and HB2151, TAG is recognized as stop codon that
terminates translation at C-terminus of foreign
proteins and allows expression of the foreign pro-
teins in a soluble form.

18.  First, the phagemid particles containing the
DNA fragment encoding a foreign protein
infect theF^strain of Escherichia coli. Upon
entering the host cell, the single-stranded
DNA (ssDNA) of phagemid particles can be
converted into replicative form (RF) of the
phagemids by the host RNA and DNA
polymerases and topoisomerase, which act
on the replication origin of minus strand
located in the IG region of the phagemids.

29. Types of phage display systems

30.  In a “type 3” vector, there is a single phage chromosome
(genome) bearing a single gene III which accepts foreign DNA
inserts andencodes a single type of pIII molecule. The foreign
peptide encoded by the insert is theoretically displayed on all
five pIII molecules on a virion (though in practice normal
proteolytic enzymes in the host bacterium often remove the
foreign peptide from some or even most copies of pIII, especially
if the foreign peptide is large). Similarly, type 8 . In a type 88
vector, the phage genome bears two genes VIII, encoding two
different types of pVIII molecule; one is ordinarily recombinant
(i.e., bears a foreign DNA insert) and the other wild-type. The
resulting virion is a mosaic, its coat comprised of both wild-type
and recombinant pVIII molecules (the former usually
predominating). This allows hybrid pVIII proteins with quite large
foreign peptides to be displayed on the virion surface, even
though the hybrid protein by itself cannot support phage
assembly. Similarly, a type 33 vector bears two genes III, one of
which is recombinant.

31.  A type 8+8 system differs from a type 88 system in that the two genes VIII
are on separate genomes. The wild-type version is on a phage (usually called
the “helper” phage), while the recombinant version is on a special kind of
plasmid called a “phagemid. Like other plasmids used in recombinant DNA
research, a phagemid carries a plasmid replication origin that allows it to
replicate normally in an E. coli host and an antibiotic resistance gene that
allows plasmid-bearing host cells to be selected. But it also carries a
filamentous phage replication origin, which is inactive until the cell is
infected with the helper phage. Then the phage replication protein acts not
only on the phage origin on the helper phage DNA but also on the phage
origin on the phagemid DNA. Two types of progeny virions are thus secreted:
particles carrying helper phage DNA and particles carrying phagemid DNA.
Both these virions, like the type 88 virions, are mosaics, whose coats are
composed of a mixture of recombinant and wild-type pVIII molecules. When
a phagemid virion infects a cell, the cell acquires the antibiotic resistance
carried by the phagemid. When a helper phage virion infects a cell, the cell
goes on to produce progeny helper virions in the normal way; the progeny
virions, unlike the original infecting virion, are not mosaic, since the helper
carries only a single gene VIII. Type 3+3 and 6+6 systems are like type 8+8
systems, except that the phagemid carries an insert-bearing recombinant
gene III or VI, respectively, rather than VIII. The recombinant pIII encoded by
a type 3+3 phagemid is usually missing the N-terminal domain, since cells
expressing this domain are resistant to superinfection by helper phage.

38.  Fab
 F(ab)2
 Fv
 ScFv
 HcAb

47.  Library A population of clones with each
clone containing one random piece of
chromsomal DNA cloned into a vector.

49.  Production of gene fragment: This phase involves
animal immunization with the desired antigen
and then isolation of B lymphocytes, mRNA
extraction and cDNA synthesis. The synthesized
cDNA contain genetic information of all
antibodies targeting various antigens and consist
of approximately10 ^9to10^ 11 lymphocyte
clones.Cloning of gene fragments in the
phagemid vectors: Genes related to the different
clones of antibodies are digested with restriction
enzymes, clone into phagemid vectors and then
display on the surface of phages.

50.  So far, a variety of phages like phages l, T4,
T7and M13 have been described for
displaying of antibody fragments. These
vectors help to the displayed antibodies to
maintain their function at the surface of
phage. But using of phages like M13 that not
destroy the bacterial cells is most
applicable.The phagemid vectors need helper
phage to package and exit from the bacterial
cells and enter to the medium.

51.  Selection of specific phages: After cloning of the
fragments into phagemid vectors, because of
diversity in antibody gene,variety of clones of
antibodies display on the surface of the phage.
Selections of specific clone that recognize the
antigen(target of interest) perform by
biopanning. Since the antibody fragments on the
surface of the phage are functional so the phage
carrying specific antibody can be isolated from
non-specific phages (due to antigen–antibody
binding properties).

52.  The panning process include: immobilization
of antigen, binding of the phages, washing
and removing non- bonded phages, elution
of the bonded phages, re-infection of the
bacterial cells to amplify the eluted phages,
purification of the recombinant phages and
re-expression of antibodies on the surface of
phage. Biopanning usually repeat 3 to 5 times
to isolate specific antibodies with high affinity
to target.

53.  Screening: Isolation of antibodies with high
affinity to target is the main aim of this step.
Screening is performed using different methods
like: immunoassay, immunocytochemistry,active
isolation of cells due to their fluorescent
properties and immunoblotting. In immunoassay,
at first antigen is coated on solid phase, phage
displaying antibody is added and then secondary
antibody that detect the surface proteins of
phages is added. After incubation and washing
the signal value and binding is measured in
appropriate wave length.

66. Screening key act

77.  Production of mAbs by phage display:
Hybridoma and phage display technology are
common techniques to obtain monoclonal
antibodies.Combination of B cell producing
monoclonal antibody with mouse myeloma
cells is the basis of hybridoma technology.
 Despite of advantage of hybridoma
technology there are some limitation: poor
immunogenicity of some targets, high
production cost,, time-consuming and need
to myeloma cells.

78.  Phage display has been used in enzymology to determine the
substrate specificity and to develop modulators of both the
active and
allostericsitesoftheenzyme(Diamond,2007;KehoeandKay,2005
;Kayetal.,2001;Benhar,2001).Themethodcanbeusedtodisplaym
utantsof 854J. Pande et al. / Biotechnology Advances 28
(2010) 849–858 enzymes to study their mechanisms of action
(Vanwetswinkel et al.,2000; Ponsard et al., 2001; Verhaert et
al., 2002). Since filamentous phage is resistant to broad
range of proteases, it has been used in identification of
substrates of various proteases (Matthews and
Wells,1993;Diamond,2007).

79.  Phage display is a powerful technique in isolation of
monoclonal antibodies with high affinity to their target.
 Epitope mapping and finding mimotopes
 Another application of phage display is to map epitope or
binding site of antigen that is involved in interaction with
anti body .Peptide phage display libraries described in
above are useful tools for identification of continuous or
linear epitopes involve in interaction with antibody.
Isolation and identification of mimotopes from peptide
phage libraries is powerful approach to improve
immunological studies in order to design and develop
vaccine candidate.Through peptide phage library and
biopanning process, phages carrying peptides mimic the
epitopes of the antigen are identified. Length of epitopes
that are recognized by antibodies is between four to six
amino acids. So, heptapeptide libraries are appropriate for
identification of all linear epitopes.

80.  Phage display has been used to identify agonists and antagonists to
probe the receptor structure and function. The peptide libraries can be
screened for binding to functionally folded extracellular domains of
receptors that contain the site for natural ligand. Selected peptides that
recognize the binding interface of the receptor can antagonize its
interaction with the natural ligand. The structural and functiona
properties of individual members of a large receptor family that bind the
same natural ligands can be characterized with affinity selected peptides
specific for each member (Koolpe et al., 2005). Phage encoded peptide
ligands have also been selected for targets like G-protein coupled
receptors, in which it is difficult to purify the
functionallyfoldedextracellular receptor domains. Antibodiesspecific for
the known receptor ligand can be used as a target to affinity select
mimotopes of the ligand from phage display library. The selected
mimotopes can be used to study the mechanism of interaction of ligand
with its receptor and allow the development of potent agonist and
antagonists (Bonetto et al., 2005). Receptor antagonists can also be
obtainedby selecting peptides thatbindtothereceptoragonistand thereby
inhibit its interaction with the receptor. An example is the invention of
antagonistic peptide binding insulin-like growth factor-1(IGF-1)
(Deshayes et al., 2002)