A SEMINAR ON PHAGE DISPLAY TECHNIQUE & APPLICATION
Dr. ARUNIMA KARKUN
PHAGE DISPLAY-TECHNIQUE AND APPLICATION INTRODUCTIONHISTORYTECHNIQUECREATION OF VECTOR AND FUSIONBINDING AND SELECTIONWASHINGELUTION AMPLIFICATION APPLICATION EPITOPE DETERMINATIONENZYMATIC APPLICATION ORGAN TARGETING GENE DELIVERYISOLATION OF ALLERGENSADVANTAGESDISADVANTAGESCONCLUSION SUMMARYREFERENCES
PHAGE DISPLAY-TECHNIQUE AND APPLICATION Bacteriophages are viruses that infect bacterial cells.
Infected cells are used as a host to replicate viruses.
It is an in vitro selection technique using a protein genetically fused to the coat protein of a bacteriophage.
E. coli phages used due to ease of culture and quick regeneration.
PHAGE DISPLAY-TECHNIQUE AND APPLICATION George P. Smith 1985 first introduced Phage display technology
Bass et al 1990 A large number of phage displayed peptide and protein libraries have been constructed.
PHAGE DISPLAY-TECHNIQUE AND APPLICATION This technique is called phage display because it involves the display of protein on the surface of Bacteriophage.In this , cloning vector used for phage display.Following steps involved in the phage display-Creation of vector and fusionBinding and selectionWashingElution Amplification TECHNIQUE5
PHAGE DISPLAY-TECHNIQUE AND APPLICATION Creation of vector and fusion Recombinant DNA technology to incorporate foreign cDNA of interest into viral DNA.
The cloned gene becoming fused with a gene for the phage coat protein.
So the protein will be displayed on outside of phage particles.TECHNIQUE6
PHAGE DISPLAY-TECHNIQUE AND APPLICATION Binding and selection Can apply standard affinity techniques to capture phage by taking advantage of displayed proteins.
Pass solutions of amplified phages over solid support with antigens or receptors bound to it.
Phages with affinity to support bind. TECHNIQUE7
PHAGE DISPLAY-TECHNIQUE AND APPLICATION Washing Unbound phages are washed away leaving only those showing affinity for the receptors.ElutionBound phages can be eluted by disrupting the protein bonding interactions. Acidic buffers, Alkaline buffers, Urea, addition of soluble ligend for receptor. Can also add host cells to infect.
PHAGE DISPLAY-TECHNIQUE AND APPLICATION Amplification Eluted phages showing specificity are used to infect new host cells for amplification.
Cycle repeated 2-3 times for stepwise selection of best binding sequence.TECHNIQUE9
PHAGE DISPLAY-TECHNIQUE AND APPLICATION
Fig. No.- 1, Steps involved in phage display technique. TECHNIQUE
PHAGE DISPLAY-TECHNIQUE AND APPLICATION
Fig. No.-2, Amplification of bacteriophage.TECHNIQUE
PHAGE DISPLAY-TECHNIQUE AND APPLICATION Phage display technique have following application-
EPITOPE DETERMINATIONENZYMATIC APPLICATION ORGAN TARGETING GENE DELIVERYISOLATION OF ALLERGENSAPPLICATION12
PHAGE DISPLAY-TECHNIQUE AND APPLICATION Epitope determinationPeptide libraries have been used to determine the epitope to which an antibody binds.
Therefore, it is possible to define the region of a protein recognized by an antibody.
Phage libraries were used to define peptide structures recognized by major histocompatibility (MHC) molecules. APPLICATION13
PHAGE DISPLAY-TECHNIQUE AND APPLICATION ENZYMATIC APPLICATION
In enzymology, it has been applied for mechanistic- based studies and to generate enzyme variants with new or improved properties.
Enzymes with a broader range of substrates, for instance, would have improved function and allow certain advantages to its host. APPLICATION14
PHAGE DISPLAY-TECHNIQUE AND APPLICATION ORGAN TARGETINGIn this technique phage libraries are injected intravenously into animals and then organs or tissues are collected and examined for phage bound to tissue-specific endothelial cell markers.
GENE DELIVERYGene delivery to mammalian cells has also been accomplished by the use of single and double- stranded phage.APPLICATION15
ISOLATION OF ALLERGENS
Fusion proteins created by the principle of linking the phage phenotype to its genetic information are covalently associated with the phage particle.
Therefore, cDNA libraries displayed on phage surface can be screened for the presence of specific clones by affinity purification. PHAGE DISPLAY-TECHNIQUE AND APPLICATION APPLICATION16
PHAGE DISPLAY-TECHNIQUE AND APPLICATION Phage clones that bind to IgE may be selected by screening and enrichment of phage libraries against serum IgE immobilized in a solid phase.
The amino acid sequence of surface-expressed allergens can be clarified by sequencing the DNA of the integrated section of the phage, as there is a physical linkage between its genotype and phenotype.APPLICATION17
PHAGE DISPLAY-TECHNIQUE AND APPLICATION 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.
PHAGE DISPLAY-TECHNIQUE AND APPLICATION 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.
PHAGE DISPLAY-TECHNIQUE AND APPLICATION Phage display allows the presentation of large peptide and protein libraries on the surface of filamentous phage, which leads to the selection of peptides and proteins, including antibodies, with high affinity and specificity to almost any target. The technology involves the introduction of exogenous peptide sequences into a location in the genome of the phage capsid proteins. The encoded peptides are expressed or "displayed" on the phage surface as a fusion product with one of the phage coat proteins.CONCLUSION20
PHAGE DISPLAY-TECHNIQUE AND APPLICATION Bacteriophages are viruses that infect bacterial cells.Infected cells are used as a host to replicate viruses.Phage display technique involves the following steps- Creation of vector and fusionBinding and selectionWashingElution Amplification Phage display technique have following application-Epitope determinationEnzymatic application Organ targeting Gene deliveryIsolation of allergens
PHAGE DISPLAY-TECHNIQUE AND APPLICATION T. A. Brown 2008 Gene cloning and DNA analysis
Some contents from net- www.mupnet.com/jocm.htm www.bio.anl.gov/antibody.html REFERENCE22