A SEMINAR ON PHAGE DISPLAY – TECHNIQUE & APPLICATION

Dr. ARUNIMA KARKUN

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PHAGE DISPLAY-TECHNIQUE AND APPLICATION

INTRODUCTIONHISTORYTECHNIQUE

•CREATION OF VECTOR AND FUSION•BINDING AND SELECTION•WASHING•ELUTION •AMPLIFICATION

APPLICATION •EPITOPE DETERMINATION•ENZYMATIC APPLICATION •ORGAN TARGETING •GENE DELIVERY•ISOLATION OF ALLERGENS

ADVANTAGESDISADVANTAGESCONCLUSION SUMMARYREFERENCES

SYNOPSIS

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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.

INTRODUCTION

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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.

HISTORY

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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 fusion•Binding and selection•Washing•Elution •Amplification

TECHNIQUE

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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.

TECHNIQUE

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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.

TECHNIQUE

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PHAGE DISPLAY-TECHNIQUE AND APPLICATION

Washing• Unbound phages are washed away leaving only those showing affinity for the receptors.Elution• Bound 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.

TECHNIQUE

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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.

TECHNIQUE

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PHAGE DISPLAY-TECHNIQUE AND APPLICATION

Fig. No.- 1, Steps involved in phage display technique.

TECHNIQUE

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PHAGE DISPLAY-TECHNIQUE AND APPLICATION

Fig. No.-2, Amplification of bacteriophage.

TECHNIQUE

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PHAGE DISPLAY-TECHNIQUE AND APPLICATION

Phage display technique have following application-

•EPITOPE DETERMINATION•ENZYMATIC APPLICATION •ORGAN TARGETING •GENE DELIVERY•ISOLATION OF ALLERGENS

APPLICATION

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PHAGE DISPLAY-TECHNIQUE AND APPLICATION

Epitope determination•Peptide 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.

APPLICATION

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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.

APPLICATION

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PHAGE DISPLAY-TECHNIQUE AND APPLICATION

ORGAN TARGETING•In 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 DELIVERY•Gene delivery to mammalian cells has also been accomplished by the use of single and double- stranded phage.

APPLICATION

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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

APPLICATION

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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.

APPLICATION

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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.

ADVANTAGES

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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.

DISADVANTAGES

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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.

CONCLUSION

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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 fusion•Binding and selection•Washing•Elution •Amplification

Phage display technique have following application-

•Epitope determination•Enzymatic application •Organ targeting •Gene delivery•Isolation of allergens

SUMMARY

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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

REFERENCE

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