13. PHAGE DISPLAY

PHAGE DISPLAY
Phage display describes a selection technique in which a library of variants of a peptide or protein are
expressed on the outside of phage virions, while the genetic material encoding each variant resides on the
inside of the corresponding virion . 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.
Protocol (simple, basic)
1. INCUBATION AND WASHING
Incubation of 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.
2. AMPLIFICATION
The eluted phage is amplified and taken through additional binding/ amplification cycles to enrich
the pool in favor of binding sequences. (3-4 rounds)
3. SEQUENCING
Individual clones are characterized by DNA sequencing. Pile up of obtained aminoacid sequences
leads to a CONSENSUS sequence or molecular LOGGO. This is done with programs such as
SEQ2LOGO
¿What is panning?
Surface Panning Procedure (Direct Target Coating)
The most straightforward method of affinity partitioning (panning) involves directly coating a plastic
surface with the target of interest (by nonspecific hydrophobic and electrostatic interaction), washing away
the excess, and passing the pool of phage over the target‐coated surface. Because of its relative simplicity,
we recommend trying the direct coating method first
Protocol (extended)
1. OVEREXPRESSION
Overexpress the recombinant protein (target) in bacterias with protein expression by introducing
the cDNA with tag in a vector and purify.
2. IMMOBILISATION OF TARGET TO A SURFACE
The target has to have affinity for the protein exposed in the surface of the phage, to capture only
those who have our protein of interest. This can be done by beads, resins of metallic ions or directly
in a plaque. This process is incubated at 4ºC overnight.
Washings are then done with Tris Buffer Solution
3. IMMOBILISATION OF PHAGES THAT PRESENT THE PROTEIN OF INTEREST TO THE SUFACE
With blocking buffer
4. WASHINGS OF NON-ADHERED PHAGES
The phages that don’t present affinity with the target molecule are eliminated with various
washings.
5. ELUTION
(of phages that are adhered). The particles of captured phages are first eluted with Tris Buffer
Solution and then used to infect E.Coli. In this step there are phases of selection in which the clones
of less affinity are eliminated.
6. CENTRIFUGATION
To obtain as the precipitate the phages
7. AMPLIFICATION
The amplification of these phages is needed so that these can be used in a new cycle of selection.
8. SEQUENCING
Instead of being amplified, the DNA of the phages can be sequenced by the bioinformatics
programs.