In 1985, Smith transformed filamentous phages through genetic engineering technology, obtained recombinant phages that could proliferate in vitro, and created phage display technology. In 1988, by fusing the N-terminus of the phage pⅢ protein with a known antigenic determinant and presenting it on the phage surface, the fusion could specifically bind to antibodies, which led to the idea of understanding antigenic epitopes through the construction of phage peptide libraries. As a technology for studying antigenic epitopes, phage display mainly uses genetic engineering technology to insert exogenous genes into phage capsid proteins, so that the polypeptides encoded by the exogenous genes can be displayed as fusion proteins, and their own structure and activity do not change. After that, multiple rounds of screening are used to select highly specific antibodies or peptides that can bind to target molecules. In 1990, a random 6-peptide library based on phage display technology was created. After years of development, phage display technology has been used in the fields of cell signal transduction, protein recognition sites, and drug development in addition to studying antigenic epitopes. The capacity of peptide libraries established based on phage display technology is as high as billions. In terms of screening peptide markers, phage display peptide libraries have the advantages of fast, relatively low cost and large library capacity. By screening phage peptides that bind to specific antigens, new antibodies can be developed or existing antibodies can be improved. Phage display peptides can also be used for vaccine development, cancer diagnosis, biosensor development, etc.

Classification of Peptide Libraries:

Peptide libraries based on phage display are mainly composed of short peptide sequences of different lengths. According to different construction methods and uses, peptide libraries are divided into different types. Overlapping peptide libraries are constructed by gradually intercepting different fragments of protein sequences to construct phage peptide libraries. They are mainly used for full-length proteins and help to identify continuous linear epitopes. The peptide library constructed by removing amino acids on both sides of the original polypeptide sequence is called a truncated peptide library. It is used to determine epitopes in a small range and helps to identify amino acid sequences related to biological activity. In the alanine peptide scanning library, alanine can replace each amino acid residue, so as to understand the role played by each amino acid. The random peptide library contains a combination of all possible arrangements of 20 kinds of amino acids. Since these peptide sequences are full of randomness, the random peptide library contains a large number of different peptides, and more peptides with specific functions or interactions can be discovered through the random peptide library. The scrambled peptide library can rearrange the original amino acid sequence, with high variability. It is usually used as a negative control to illustrate the importance of amino acid sequences arranged in a specific order for the structure and function of the peptide.

The peptide library composed of amino acids arranged linearly is called a linear peptide library. It has a simple structure, is easy to synthesize and characterize, and can adjust the sequence and length of amino acids as needed. It is mainly used to study the interaction between proteins and the screening of antigenic epitopes. Some chemical reactions, such as amide bond cyclization, can transform the linear peptide library into a polypeptide chain library with a cyclic structure, that is, a cyclic peptide library. Since cyclization can promote the binding of intramolecular hydrogen bonds, reduce the ability of external hydrogen bonds in molecules, improve protein resistance, and prolong half-life, the stability of the cyclic peptide library is stronger. Since the surface area of cyclic peptides is relatively large, the probability of binding to receptors is increased, and it can even target target proteins without binding pockets. Different cyclization modes and cyclization binding sites can produce a large number of cyclic peptide molecules, and the possibility of discovering new ligands is also increased. Some cyclic peptides have strong cell penetration and oral properties, and have potential application value in the development of therapeutic drugs.

Synthesis and Screening of Peptide Libraries:

The basic principle of phage peptide library construction is to create a library containing a large number of peptide sequences of different lengths, amino acid compositions and structures, and then identify peptides with specific functions through random peptide library screening. The synthesis steps include the generation of random peptide sequences, the synthesis of peptides, and their display or expression on specific carriers. When constructing a 12-peptide library, an algorithm is first used to generate a large number of random 12-peptide sequences. In order to ensure the diversity of the library, these sequences can cover all possible amino acid combinations. After that, the peptide is synthesized. The most commonly used method is solid phase synthesis. In order to prevent side reactions, all amino acids involved in the reaction are protected by protective groups. Common protective groups include Fmoc and BOC. First, the amino acid at the C-terminus is connected to the solid phase carrier and its protective group is removed. The remaining amino acids are added in sequence, and the steps of eluting the protective group, connecting and washing are repeated until a complete 12-peptide sequence is synthesized. The generated 12-peptide sequence is fused to the N-terminus of the M13 phage pIII protein, and then screened and identified by phage display technology.

Fig. 1 Schematic diagram of peptide library synthesis

Solid phase screening and liquid phase screening are two commonly used random peptide library screening methods. Among them, solid phase screening will fix the target molecule on a solid phase carrier, such as a 96-well plate, and then incubate the peptide library with the fixed target molecule, then remove the unbound peptide by washing, and finally elute the bound peptide from the solid phase carrier for subsequent analysis. The operation of solid phase screening is relatively simple, and there is no need to label the antigen. Multiple samples can be processed at the same time, which is suitable for large-scale screening, but it is also prone to non-specific binding. Liquid phase screening mainly uses magnetic beads as the screening platform. After the target antigen is combined with the magnetic beads, it is incubated with the peptide library, and then the magnetic beads bound to the peptide are separated for subsequent analysis and identification. Liquid phase screening can reduce the occurrence of non-specific binding, but it requires special instruments and equipment to operate. It is suitable for screening with reduced antigen concentration gradients and screening of high-affinity peptides.

TekBiotech is committed to providing customers with high-quality peptide library construction technology services. We have rich project experience and insights in peptide library construction. After years of development, TekBiotech has established a complete peptide library construction system, including the M13 phage display system, M13KE phage display system, T7 phage display system, etc., which rely on M13 helper phage. It can provide customers with high-quality linear peptide libraries (including but not limited to 6-peptide library, 7-peptide library, 12-peptide library, 15-peptide library) and cyclic peptide libraries (cyclic 6-peptide library, cyclic 7-peptide library, cyclic 10-peptide library, etc.) and other types of peptide library construction services. The M13 phage peptide library established by TekBiotech has a library capacity of up to 10^8 and a titer of up to 10^13 phage display peptide particles/ml. The T7 phage peptide library has a library capacity of up to 10^8 and a titer of up to 10^11 phage display peptide particles/ml, which is sufficient to support customers' subsequent screening of targeted peptides for various targets and meet downstream experimental needs. TekBiotech can provide one-stop technical services ranging from peptide gene library design and synthesis, peptide library construction to supporting peptide library screening, affinity verification, in vitro cell verification, etc. Customers only need to provide specific project requirements, and TechBio scientists will design the best library construction method and phage system according to the customer's project requirements to meet the customer's project needs.