Phage Display and it's Application

Introduction

T phage
structure of a T Phage

Phage display is a molecular biology techniques that involve an 
in vitro screening technique for identifying ligands for proteins and other macromolecules. In this technique, a gene encoding a protein of interest is inserted into a phage coat protein gene, causing the phage to "display" the protein on its outside while containing the gene for the protein on its inside, resulting in a connection between genotype and phenotype. This technique has evolved for years and years with a lot of improvement to reach the point its one of the important technique in protein engineering.
in this technique, in vitro selection can be done by screening and amplifying large libraries of protein, which is analogous to natural selection. Phage display is also an effective way for producing large amounts of peptides, proteins and antibodiesThe most common bacteriophages used in phage display are E.coli filamentous bacteriophages (f1, fd, M13) , though T4 , T7, and λ phage have also been used.
this technique was first developed by G. Smith in 1985 as a method of presenting polypeptides on the surface of lysogenic filamentous bacteriophages.

Applications of Phage Display

Nowadays, phage display as a rapidly developing technology has been used in a wide range of applications in different research areas including epitope mapping, identification of new receptors & ligands, in vitro protein evolution, drug discovery, and so on. Some of the most successful applications of phage display are explained in the following sections.

1 Epitope mapping and mimicking

Upon encountering antigen, host humoral immunity activates and triggers production of antibodies which directed against foreign protein epitopes. Knowledge of these protein epitopes is pivotal in understanding the pathogenesis of pathogen infections and in developing diagnostic reagents, therapeutic antibodies, and effective vaccines.

Peptide phage display libraries are useful tools for identification of continuous or linear epitopes involving in interaction with antibody. Isolation and identification of mimotopes from peptide phage libraries is powerful approach to improve immunological studies in order to design and develop vaccine candidate .

2 Identification of new receptors & ligands

Phage display method using either gene-specific libraries, or random peptide libraries, provides a powerful technique for an approach to epitope identification. The technique can identify amino acids on protein antigens that are critical of antibody binding.

The random peptide sequence was displayed on the surface of the phage to obtain the phage display polypeptide library. Polypeptides identifying specific cells were obtained by differential screening using cells as screening targets. By studying the polypeptide sequence, we can further to obtain the receptor protein expressed specifically on the cell surface.

3 Protein-protein interaction studies

Protein–protein interactions mediate essentially all biological processes. A detailed understanding of these interactions is thus a major goal of modern biological chemistry. Phage display is a potent and versatile method for studying protein-protein interaction. It can be applied to a wide range of protein interaction partners and used in a number of applications, especially in mapping intracellular interactions of the distinct protein domain.

The polypeptide library displayed by bacteriophages is composed of random short peptide sequences of specific length. Short peptide sequences can be obtained by affinity screening for the random library by target proteins (such as receptors). Sequence analysis and synthesis of corresponding short peptides, and then we can study the interaction between two proteins.

4 Recombinant Antibody Production

Recombinant antibodies are useful tools for therapy, diagnosis and research. With major developments in molecular biology, numerous display technologies have been successfully introduced for recombinant antibody production. Even so, antibody phage display still remains the gold standard for recombinant antibody production. Its success is mainly attributed to the robust nature of phage particles allowing for automation and adaptation to modifications.

a. Production of gene fragment: This phase involves animal immunization with the desired antigen and then isolation of B lymphocytes, mRNA extraction and cDNA synthesis. The synthesized cDNA contain genetic information of all antibodies targeting various antigens and consist of approximately 109 to 1011 lymphocyte clones.

b. Cloning of gene fragments in the phagemid vectors: Genes related to the different clones of antibodies are digested with restriction enzymes, clone into phagemid vectors and then display on the surface of phages. Sequence diversity of the fragments at this step leads to optimum isolation of antibodies in the later steps. The phagemid vectors need helper phage to package and exit from the bacterial cells and enter to the medium.

c. Selection of specific phages: After cloning of the fragments into phagemid vectors, variety of clones of antibodies display on the surface of the phage. Selections of specific clone that recognize the antigen (target of interest) perform by biopanning. Then the phage carrying specific antibody can be isolated from non-specific phages due to antigen–antibody binding properties.

d. Screening: Isolation of antibodies with high affinity to target is the main aim of this step. Screening is performed using different methods like: immunoassay, immunocytochemistry, active isolation of cells due to their fluorescent properties and immunoblotting.

You can construction phage display library by yourself or choose some phages display companies to do it. Also you can by a pre-made library directly. If you already had a pre-made library, you can just start at selection. Pre-made antibody libraries represent a naïve antibody repertoire which consists billons of different antibody scFv or fad genes. You should select the ones that actually recognize your antigen. If the repertoire is diverse enough, there are antibodies against your antigen present, and you just have to enrich them.

5 Protein directed evolution

Phage display technology as a selection based system is an attractive method for evolution of new biological drugs. Directional transformation protein refers to use cassette mutation, error-prone PCR method to mutant protein or a particular cod sequence structure domain. Proteins or structural domain mutations present library will be display in phage surface. Then we can obtain the required have directional change of phage clones by affinity screening. The primary structure can be derived from the sequence of DNA that can be used to screen more cytokines receptors ability, new enzyme inhibitors, DNA transcription factors in combination with the new sites, new cytokine antagonists, new enzymes and enhance biological active protein.

6 Drug discovery

Peptides as biologically active molecules in hormones, neurotransmitters, cytokines, antigens, and growth factors are involved in a wide variety of biological processes. So, peptides are extensively used as therapeutic and diagnostic agents in the medical fields such as oncology, endocrinology, urology, and obstetrics. The peptide phage libraries with presenting a huge number of different peptides mimicking the genuine epitopes play a key role in the development of new therapeutic peptides. Until now, several peptide drugs have been developed using phage display technology.

7 In vitro diagnostic

At present, phage display library technology is broadly employed to examine host-pathogen interaction, development of disease diagnostic markers, and identification of vaccine candidates and novel antibodies against pathogenic targets.

The cell-surface antigens in pathogens as molecular binding sites are suitable targets for vaccine development, since these antigens can affect the bacterial division, replication, and virulence of pathogens. There are two distinct strategies for application of phage display technology in infectious diseases area: In the pathogen-targeted phage display, molecular targets such as cell replication enzymes or host-pathogen virulence factors are targeted for screening; while in the cell-based screening method, bacterial whole cell is employed as target for screening. The cell-based screening with live pathogens can target the native cell surface proteins. This screening method is suitable when target antigen is unstable under immobilization conditions.

read more about procedure of phage display here

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