Peptide Phage Display Library Construction

Alpha Lifetech Inc. can offer native, semi-synthesized, and fully synthesized peptide libraries based on phage display technology. Our experts have extensive experience in library construction, including chemical peptide libraries, phage display peptide libraries (such as substrate peptide libraries), antibody libraries, and related libraries.


Peptide Phage Display Library Construction


The basic principle of phage peptide library is to insert random peptide segments into phage coat proteins to form fusion proteins and display them, and make use of the characteristics of phage replication to obtain multiple copies of different recombinant phages, providing a favorable tool for different studies.

Randomness refers to the random sequence of the gene encoding the peptide, which is usually derived from the degradation of the oligonucleotide chain synthesized by chemical methods. Sometimes, the entire sequence of a gene (such as antibody or viral genome) can be randomly inserted into the phage to form a random peptide library of a gene, generally called antibody library or target gene library. The inserted peptide segments are generally displayed on the phage surface in a linear form, ranging from several peptides to several peptides. At present, the peptide library has developed from the initial 6 peptides and 7 peptides to 9 peptides, 15 peptides, 38 peptides, and 40 peptides. The longer the inserted fragment, the larger the storage capacity required to ensure diversity. It has been envisaged to construct peptide libraries of 15 to 100 amino acid residues to contain some of the secondary structures found in natural epitopes. However, the construction of a phage peptide library still depends on the transformation of DNA, and the most effective electric conversion rate can only reach 109~1010 DNA at present. Moreover, it is difficult to enrich the required phage recombinant peptide from the expansion library whose diversity is higher than 1012. Third, it is difficult to enrich the phage recombinant peptide after the phage expresses the gene inserted in the phage. It is often displayed as a linear peptide on the bacterial coat protein, and the single structure of this peptide segment limits its application.

Phage display of peptides is widely used for the development of peptide ligands and epitope mapping. The procedure involves 2 - 4 iterative rounds of affinity selection and phage amplification followed by an optional ELISA-based screen and sequencing of several dozens of positive clones. A panel of peptides is synthesized, and their binding to the protein target or biological activity is tested. Compared to the entirely artificial peptide library, polypeptide libraries based on the phage display technique are different since phage particles can carry peptides with hundreds of amino acids.


Fig 1 Phage display methodology


Screening of Peptide Phage Display Library


How to select specific recombinant phage from phage peptide library is the key to peptide library technology. At present, a method called "biological panning" is commonly used. According to the different states of the target molecule (antibody or receptor), the screening methods can be divided into solid-phase screening, liquid-phase screening, and so on. The remaining operation procedures are the same: the target molecule is coated, the non-specific site is closed, the phage peptide library is added for co-incubation, and the unbound phage is washed away. Subsequently, specific bound phages with competitive receptors or pickling adsorption infect logarithmic host bacteria, multiply and expand, and then undergo the next screening round. After 3-5 rounds of adsorption-elution-amplification, phages presenting specific peptides were highly enriched, and eventually, peptides/proteins with specific binding properties could be obtained, and gene cloning could be achieved.

The most obvious advantage of biological screening technology is that the DNA sequence of the target molecule can be obtained in vitro (outside bacteria) for the study of foreign peptides.


Fig 2 Panning phage-displayed random peptide libraries with antibody-coated wells


Application of Peptide Phage Display Library


Peptide libraries often have been used to explore protein-protein interactions, including antigen-antibody interactions and substrate-enzyme interactions (substrate finding), and to map intracellular protein networks for high throughput and high content screening. Combined with next-generation sequencing (NGS) technology, peptide libraries can be a very powerful tool that makes it possible to screen substrate sequences of enzymes from large and diverse collections of randomized sequences without any initial substrate data. The sensitivity and versatility of this technique have been clearly established through the discrimination of the substrate specificity of closely related proteases or protein tyrosine kinases.

Other Applications:

-- Antigen Epitope Study

Phage peptide library technology can screen recombinant phage peptide sequences that can bind to specific antibodies by exposing antibodies to peptide libraries, which makes up for the shortage of synthetic peptide screening. In the study of antigen-antibody recognition, epitopes that can bind to antibodies can be directly screened from random peptide libraries, which are either linear epitopes or simulated conformational epitopes, and the amino acid sequence of proteins need not be determined in advance. Although it may sometimes differ from natural epitopes in amino acid sequence, it can simulate the binding properties of natural ligands and can induce antibodies that cross-react with natural epitopes after immunizing animals, and both competition and inhibition tests have shown that it can play the role of natural epitopes.

-- Immunodiagnostic Study

The serum of patients with infectious diseases contains various antibodies against the antigen. However only when the natural antigen is a microorganism or has been cloned and expressed is it easy to obtain a pure antigen to identify the specific antibody. The phage random peptide library technology can be used as antigen simulation for the immunodiagnosis of clinical diseases when some natural antigens are unknown or not readily available.

-- Genetic Vaccine Research

The biggest advantage of using a peptide library is that it does not require any structure of the target protein. Only its antibodies or receptors can be used to capture the small ligand in the phage peptide library. They can be multiplied in large numbers, which provides great convenience for the design and production of vaccines. Experiments have shown that filamentous phages have good immunogenicity in a variety of animal systems and can be used as carriers to synthesize complete antigens with antigen mimics peptides, causing immune responses similar to those caused by natural antigens in mice. The generated antibodies can react with natural antigens. Phage polypeptides can be screened and identified as vaccines by detecting the levels of antibodies produced by immune mice.

-- Drug Screening and Development Research

 Due to the lack of understanding of the relationship between protein structure and function in traditional drug development, existing proteins can only be modified through large-scale site-specific mutation and time-consuming cloning, expression, purification, and screening of single target proteins. With phage display peptide technology, small peptides or novel proteins with strong affinity and specificity to target molecules can be easily and quickly obtained by using target molecules as receptors and screening ligands in natural proteins or functional framework phage display libraries, which can be used as drug candidates. At present, this technology has been widely used in the research and development of drugs in cancer, AIDS, cardiovascular diseases, tissue and organ transplantation, neurological diseases, and other fields, and a large number of receptor antagonists and enzyme inhibitors have been screened.


The Features of Alpha Lifetech Inc.'s Peptide Phage Display Library Construction Service


-- Excellent for studying protein interactions

-- Excellent diversity: 108 - 1012

-- Excellent for high throughput and high content screening

-- Excellent for Substrate Screening


Here, Alpha Lifetech Inc. uses phagemid as a delivery system because phagemids can be modified and contain both phage and bacterial origins of replication, a phage packaging signal, a selectable marker gene, and the gene of the chosen coat protein for fusion. Its use allows easy preparation and maintenance of the vector, high yield of dsDNA, and better transformation rates. Moreover, the diversity of a randomized peptide library can reach a level of 1012, a very large and useful pool for substrate screening.

Alpha Lifetech Inc. has a mature peptide phage display platform. Our high-quality libraries may achieve greater capacity, density, and proper orientation compared to ordinary display libraries, which furthers the examination of numerous protein candidates. If you are looking for a recombinant antibody production service or a peptide scanning library and would like more information on this program, please feel free to contact us.