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 phage peptide library 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 through phage peptide library screening, 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.

Principles and Methods of Peptide Library Screening:

Peptide library screening mainly uses high-throughput technology to quickly screen out peptide sequences with specific biological activities from a large number of peptide sequences. The basic principle is to screen out peptide sequences with special structures or biological activities from a peptide library containing a large number of small peptides of specific lengths and different sequences. Peptide library screening is widely used in drug development, such as the development of new tumor therapeutic drugs, biological antibodies and anti-infective drugs.

When constructing a peptide library, an algorithm is first used to generate a large number of random 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 the complete peptide sequence is synthesized. Through the interaction with the target protein, targeted peptide screening is carried out to screen the peptide sequence that binds to the target protein from the peptide library. There are many methods for screening peptide libraries, such as affinity screening and ligand imprinting screening. Finally, the screened peptide sequences are verified and characterized to determine their binding ability and specificity with the target protein.

Fig. 1 Schematic diagram of peptide library synthesis

Two Methods for Peptide Library Screening:

The commonly used method for targeted peptide screening in vivo is the animal in vivo screening system, which injects or feeds animals with drugs containing peptide libraries, and then observes the animal's response to the drugs, and then screens out peptides with specific biological activities. The in vivo screening system can simulate the real environment of peptides in organisms, so as to screen targeted peptides. However, the operation is relatively complicated and the cycle is long. There are many factors to consider, such as animal ethics, drug metabolism, immune response, etc.

The blue-white screening system is not directly used for the screening of peptide libraries, but can be used to screen bacteria containing recombinant plasmids with exogenous DNA inserted. The blue-white system uses genetic engineering technology to insert reporter genes, such as the lacZ gene, into phage vectors. When the exogenous gene is inserted and can be expressed, the expression of the reporter gene will be disturbed, and white colonies will be formed in the host bacteria. When the inserted exogenous gene is not correctly expressed, the expression of the reporter gene will not be disturbed, and blue colonies will be formed in the host bacteria. The blue-white system is conducive to the rapid screening of phages containing exogenous genes. It can directly determine whether the clone contains the target sequence by color change, and the operation is relatively simple.

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, M13 KE 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 TechBio 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 TekBiotech 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.