M13 Phage Display Peptide Library Construction Service

Based on phage display technology, Alpha Lifetech, using M13 phage as the carrier, provides multi-type peptide synthesis service, peptide library construction and screening services, including peptide library, antibody library, etc. The peptide library of provides is rich in different peptide types, including linear heptapeptide, cyclic heptapeptide, dodecapeptide, linear nonapeptide, etc.

Bacteriophage, often called phage, is a virus that infects bacteria, including M13, T7, T4, λ and many other types of phage, among which M13 is the most widely used in phage display technology. Phage M13 belongs to the filamentous phage family, collectively known as Ff phage, and infects only strains of E. coli that express fimbriae. Its genome is a single-stranded DNA with a length of 6407 bp, encoding 11 proteins, of which 5 are structural proteins (PⅧ, pⅢ, pVI, PⅦ and PⅨ), and the remaining 6 are involved in the replication and assembly of phages.

M13 Phage Display

The core principle of M13 phage display technology is to insert the gene encoding the target polypeptide or protein into the M13 phage genome, so that the phage can express and display these foreign proteins after infecting the host bacteria. These exogenous proteins or peptides are usually fused to the C-terminal or N-terminal of a phage coat protein such as pIII or PVIII. During phage replication, these fusion proteins are displayed on the phage surface, forming a display library. In the process of phage display, a very important part is phage library construction, which needs to introduce the library containing antibody DNA sequences or other foreign DNA fragments into the phage to form a phage display library displaying various foreign peptides, then can be used in a variety of phage antibody library generation.

In M13 phage display technology, pⅢ and PⅧ are the two most commonly used display proteins. The pⅢ protein consists of 406 amino acids, three to five copies of which are distributed on one end of the phage particle, and foreign proteins or peptides can be inserted into the flexible junction area at the N end. The PⅧ protein is mainly distributed on both sides of the phage particle, and each phage contains about 2700 copies.

The main advantage of the pⅢ system is that there is no strict requirement on the size of foreign proteins displayed, and it is suitable for displaying proteins with larger molecular weights.

The PⅧ system is suitable for screening low-affinity ligands due to its large protein copy number, but it is only suitable for displaying foreign short peptides, which can affect the virus packaging and cannot form functional phages.

M13 phage display technology has a wide range of application prospects and important scientific value, and plays an important role in antibody drug development, protein interaction research, drug discovery, diagnostic reagent development and biosensing analysis.

Fig 1: Cartoon of an M13 bacteriophage virion, three types of chemically reactive groups, and primary structure of the major capsid protein, pVIII. (Reference source: Expanding the chemical diversity of M13 bacteriophage)

m13 Phage Display Peptide Library Construction Process

Fig.2 M13 Phage Display Peptide Library Constrution Service

Peptide Synthesis Service

A peptide library is a collection of many peptides with different sequences, usually obtained by chemical synthesis or biotechnology, and used in a variety of research and applications, such as medicines screening, vaccine development, protein interaction studies, etc. Alpha Lifetech provides one-stop services from peptide synthesis to phage peptide library construction to phage peptide library screening. There are two main peptide synthesis techniques available at Alpha Lifetech: Solid Phase Peptide Synthesis (SPPS) and Liquid Phase Peptide Synthesis (LPPS). Based on these two technologies, Alpha Lifetech aims to provide its customers with high quality peptide synthesis service.

Solid-phase synthesis

The peptide synthesis process is carried out on an insoluble resin, and the peptide chain is lengthened by gradually adding amino acids. The advantage of this method is that high throughput synthesis can be achieved, that is, a large number of peptides can be synthesized simultaneously. Suitable for rapid synthesis of short chain peptides. In addition, since the reaction is carried out on a solid surface, unreacted reagents and by-products can be removed by a simple washing step, simplifying the purification process.

Liquid-phase synthesis

Liquid phase synthesis refers to the gradual connection of amino acids in solution through a chemical reaction, usually involving the protection of amino acids and coupled reagents mixed together. Although this method is more complex and has a longer production cycle than the solid-phase synthesis method, it can be used for the synthesis of longer and complex peptides.

In the process of peptide synthesis, it is first necessary to protect the α-amino group and side chain functional group of amino acids to prevent unexpected chemical reactions during the reaction. When all the amino acids are connected, the protective group needs to be removed to make it active and reactive, so that it can undergo a condensation reaction with the next amino acid to form a new peptide bond. Depending on the length of the desired polypeptide coding sequence, the condensation reaction is repeated to gradually expand the length of the polypeptide chain. After completing peptide synthesis, the protective groups must be removed to restore the free functional groups of normal amino acids. The resulting polypeptides are further purified to remove unreacted amino acids, by-products, and other impurities. Common purification methods include chromatographic techniques (such as reverse phase chromatography, gel filtration, etc.). After purification, the structure type can be analyzed and confirmed by means of mass spectrometry and nuclear magnetic resonance. This step is essential to ensure the quality and purity of the peptide.

Phage Display Peptide Library Construction Workflow

Process	Service Content	Timeline
1. Design The Target Peptide	According to the research requirements, a peptide segment with a specific function or structure is designed. This peptide segment will serve as a template in the library for screening or preparing proteins with similar functions or structures.	1 day
2. Synthetic Peptide	Amino acids are the building blocks of peptide chains and are selected and prepared as needed. Synthesis usually begins at the C-terminal (carboxyl terminal) and amino acids are gradually added until a complete peptide chain is formed.	1 week
3. Peptide Modification	According to the research requirements, the synthesized peptides were chemically modified. There are many modification methods, such as lysine acylation, glycosylation, phosphorylation, etc. The modified peptide segment will have more functional groups, which will be helpful for subsequent screening and application.	1 week
4. Peptide Library Construction	The modified peptide segment was assembled according to a certain format to form a peptide library.	1 week