Phage display lecture 1 Flashcards
Phage Display Technology
An in vitro selection method that allows isolation of
antigen-specific antibodies from large collections of
molecules
Recombinant antibodies
monoclonal antibodies generated using recombinant DNA technology. These antibodies are produced by inserting the gene encoding the antibody of interest into an expression system, such as bacteria, yeast, or mammalian cells.
Antibody fragments can be expressed in bacteria
(and be displayed on phage!!)
Genes encoding antibodies isolated from B cells
a) Sequences from immunized subjects
i. Small libraries sufficient
ii. Only useful for limited set of target antigens
b) Sequences from non-immune subjects
i. Requires very large libraries, In non-immune subjects the B cells have not been selectively expanded in response to a specific antigen. Therefore, the repertoire of B cell receptors is much more diverse and includes many antibodies with low affinity or irrelevant specificities. To identify high-affinity antibodies against a specific target from this diverse pool, it is necessary to screen very large libraries of sequences to find those rare antibodies that bind effectively to the target antigen.
ii. Useful for any target antigen
Synthetic genes encoding antibodies and carrying
diversity in CDR
i. Highly functional molecules
ii. Non-natural diversity
What is a (filamentous) phage?
- Virus that infects bacteria
- Long, thin and flexible particles that contain closed circular ssDNA
- Simple structure:
- Major coat protein: pVIII
- Minor coat proteins: pIII, pVI, pVII and pIX
Phage life cycle
- pIII binds to E. coli pilus
- Pilus retracts
- Phage ssDNA enters bacterial cell and is converted to dsDNA (replivative form)
by host polymerases - Gene expression initiated and phage proteins accumulate
- Assembly and secretion of new phage
Display of fusion-ligand usually on…
pIII or pVIII.
Can any protein be displayed?
No! (e.g. full length antibodies)
phagemid
A phagemid is a hybrid of phage and plasmid vectors. It contains;
i) A plasmid replication origin
–> replicate normally in an E. coli host.
ii) Antibiotic resistance gene–> allows selection
iii) M13 phage replication origin including the packing signal, that is inactive until the cell has become infected with a helper phage.
iv) Multiple cloning sites –>to insert your protein – will be displayed on mostly p3.
v) The gene sequence for p3
vi) Lacks all the other genes required for generating a complete phage, i.e.–> will need help to generate phage particles displaying your protein.
Helper phage
a phage derivative. Contains a slightly defective origin of replication, that reduces the packing efficiency of its otherwise complete phage genome.But it can supply ALL the structural proteins required for generating a complete phage.
How do you get the antibody fragment on the surface of the phage?
Steps to Display Antibody Fragments on Phage
Preparation of Antibody Fragment Genes:
Antibody Fragment Types: Common fragments used include single-chain variable fragments (scFv) or Fab fragments. The genes encoding these fragments are synthesized or amplified from B cells.
Gene Library Creation: A diverse library of genes encoding different antibody fragments is prepared.
Vector Construction:
Phagemid Vector: The genes encoding the antibody fragments are cloned into a phagemid vector, which is a plasmid that contains the necessary elements for phage display.
Fusion Protein: The gene for the antibody fragment is fused to a gene encoding a phage coat protein, typically the gene for the pIII or pVIII coat protein of M13 bacteriophage.
Transformation and Phage Infection:
Bacterial Transformation: The recombinant phagemid vector is introduced into a host bacterium (usually E. coli).
Phage Infection: The transformed bacteria are infected with helper phages. Helper phages provide the necessary phage proteins for assembly but are deficient in packaging signals, ensuring that the phagemid is preferentially packaged into the phage particles.
Phage Assembly and Display:
Assembly: Inside the bacteria, the phages are assembled, incorporating the fusion proteins into their coat.
Phage Display: The assembled phage particles are released from the bacteria. Each phage displays the antibody fragment on its surface as part of the coat protein.
Selection (Biopanning):
Antigen Binding: The phage library is exposed to an immobilized antigen. Phages displaying antibody fragments with affinity for the antigen will bind.
Washing: Unbound phages are washed away, leaving only phages with bound antibody fragments.
Elution: Bound phages are eluted, often by using a change in pH or competitive binding with a free antigen.
Amplification and Enrichment:
Infection of Bacteria: The eluted phages are used to infect fresh bacteria, which then produce more phages displaying the selected antibody fragments.
Repeating Cycles: The process of selection and amplification (biopanning) is repeated several times to enrich the phage population for high-affinity binders
Phage display cycle
- Immobilize antigen
- Add phage library
- Wash away non-binding phages
- Elute bound phages and infect E.coli
- Amplify binding phages
- Repeat step 1-5 until sufficient enrichment
- Screen for desired properties
Display and selection technologies
- phage display (most commonly used)
- bacterial display
- yeast display
- ribosomal display
- water-in-oil emulsion display
- etc
All these techniques allow display and
selection of variant members of a population
and retain a link between
functional and genetic information
i.e. a link between genotype and phenotype
how to find the
variants with the best
properties?
By modifying the
selection conditions
(e.g. antigen concentration/addition
of competitors etc.
Ab Properties that can
be selected for in phage display:
- binding (specificity)
- affinity
- stability
- catalysis
