Phage display technique

                           

                        Phage display technique

Ø Phage display is a molecular technique that allows the expression of foreign polypeptide or peptide on their surface of phage particle protein-protein, protein-peptide, protein-DNA interaction can be studied.

Ø This method was described by George smith 1985

Ø In this technique, the DNA encoding the protein of interest is fused with gene encoding out of the proteins that forms a viral coat protein.

Ø Genetic engineering technique are used to inert the foreign DNA fragments into a suitable phage coat protein gene.

Ø Phage display involved the use of filamentous phage such as Fd, F1, M13 where the foreign gene was incorporated into gene specifying a minor coat protein.

Ø Filamentous phage M13 is the most popular choice for phage display.

Ø M13 is a filamentous phage contains 6.4kb ss circular DNA

Ø M13 enters E. coli through the bacterial sex pilus, a protein appendage the permits the transfer of DNA between bacteria.

Ø The ss DNA in the virus particle (called the (+) and (-) is replicated through an intermediate circular double stranded replicative form containing (+) and (-) strands)

Ø Only the (+) strand is packaged into new virus particles.

Ø M13 is preferred since it is non-lytic and does not destroy the host bacteria during phage production.

Ø Instead phage particles are secreted through the bacterial cell enveloped.

Ø The absence of cell debris simple’s purification of the phage.

Ø M13 phage particle consists of ss DNA molecule surrounded by a protein coat.

Ø The coat consists of major coat protein (PVII) and minor coat protein (PIII)(PIV) (PVII and PIX)

Ø At end of the particle are five copies each of the two minor coat proteins, PIX and PVII and at the other end five copies each of PIII and PVI.

Ø The minor coat protein PIII is located on the of the phage, which is responsible for attaching the phage to its bacterial host during infection.

Ø Gene encoding the minor coat protein PIII is most commonly used for recombinant formation with the DNA encoding the foreign target peptide or polypeptide.

Ø E. coli transferred with the recombinant DNA molecule produce phage particles that display peptide or polypeptide as fusion protein with endogenous PIII protein on the surface of the phage particles.

Ø There are two main method in which phage display can be used to study protein interaction.

Ø In one method, test protein is displayed and its interaction caught with series of purified protein or protein fragments of known function.

Ø In the second method, a phage display library is prepared.

Ø The library is made up of many recombinant phages each displaying a different protein.

Ø These libraries can be prepared by cloning a mixture of cDNA`s from particular tissue by cloning genomic DNA fragments.   

Advantages of Phage display

1.    They are more easily clone and Screening.

2.    They do not require animal or cell culture.

3.    They occur stable genetic sources.

4.    They can genetically manipulate.

5.    Purification easy.

6.    Cheap, simple, rapid and required non special equipment.

7.    They are useful as immunological agent.