Phage Display

Phage Display Cycle

One of the laboratory techniques employed in studying different protein interactions is Phage Display. With this in vitro screening method, protein ligands and macromolecules can be easily identified and interactions between protein and protein, peptide and protein, & DNA and protein can be studied further.

History of Phage Display

The first described instance of Phage Display occurred in 1985, when George P. Smith fused a peptide with a gene III from a filamentous phage. He filed a patent detailing the process of generating phage display libraries in the same year. Eventually, further development of Phage Display technology led by different groups from the MRC Laboratory of Molecular Biology, as well as from The Scripps Research Institute, led to the possibility of displaying proteins for the purpose of therapeutic protein engineering. The technique has been continuously improved to screen and amplify huge collections of proteins showing the connection of phenotype and genotype better.

Structure

A filamentous phage has a diameter of around 6.5 nanometers, with a length that depends on the size of its genome. It comes from a huge family of bacterial viruses that also infect other forms of bacteria. It contains a small genome with an intergenic region containing the necessary sequences for the replication and encapsidation of DNA.

A phage particle consists of five coat proteins. The particle has a hollow tube that houses so many copies of the primary coat protein. There are also binding interactions between the adjacent subunits’ hydrophobic midsections. One end of the particle is blunt, and the other is sharp. The blunt end contains plenty of copies of the two tiniest ribosomally translated proteins, while the sharp end contains around only 5 copies of the pIII and pVI genes, which are necessary for the detachment of the phage from the cell membrane.

How it works

Phage Display is a method wherein a library of phage particles that express very diverse peptides is generated. The objective is to choose those that will bind a desired target; the target can be a protein, a peptide, or a piece of DNA.

The most often used vector to build a random peptide display is the filamentous phage M13. In this display, the DNA which encodes the peptide or protein of interest is integrated into the pIII or pVI gene. To make sure that the fragments are completely inserted into the three possible reading frames, multiple cloning sites are sometimes employed, allowing the proper translation of the cDNA in its correct frame. The DNA hybrid and the phage gene are then put inside E. coli bacterial cells. Examples of these bacterial cells include XL1-Blue E. coli, SS320, TG1, and ER2738. The peptide or protein of interest is eventually expressed in either the minor or major coat protein

If another kind of vector is used, for example, a phagemid vector or simplified display construct vector, a helper phage must infect the E. coli cells; otherwise, the phage particles will not be separated from the E. coli cells. A helper phage activates the packaging of the phage DNA and assembles the mature virions with their corresponding protein fragments, which are included in the outer coating of the minor or major protein coat.

The generated phage library is then screened by addition into a microtiter plate containing immobilised target proteins or DNA sequences. Phages displaying a protein that bind to one target will remain, while the other phages can be discarded through washing. The remaining phage particles can be used to multiply the phage by infecting them into bacteria, thus increasing the diversity of the peptide display library.

Applications

The fast isolation of particular ligands through phage display has a wide variety of applications like epitope mapping, analyzing different protein interactions, vaccine development, drug design, and therapeutic target validation. Phage display is also used to pick inhibitors for the active and allosteric sites of G-protein binding modulatory peptides, enzymes, and receptor antagonists and agonists.

Determining the proper protein partners can be useful to identify the functions of various proteins. For drug discovery and design, Phage Display is employed in protein engineering or in vitro protein evolution. Therapeutic targeting with phage display is also primarily used to diagnose and determine tumour antigens, which is useful for cancer research.

Antibody Phage Display significantly improved the discovery and development of antibody drugs. Phage display for antibody libraries paved the way for rapid vaccine design and therapy. These libraries are used to learn more about the human immune system and to create human antibodies in vitro with the use of diverse synthetic substances.

Phage Display can be used in conjunction with other techniques, and with enough support and studies, more applications for it can be discovered.
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