Peptide Synthesis and Antigen Preparation

The selection of antigens is critical because it can affect the specificity, and affinity of antibodies. Common antigens include naturally purified antigens, recombinant protein antigens, and artificial antigens, among which artificial antigens include small molecule antigens and haptens.

The basic principle of small molecule antigen production is to stimulate the body's immune system and induce the body to produce corresponding antibodies by immunizing against small molecule antigens (compounds with molecular weight <1000).

Haptens (incomplete antigens) are small molecules that cannot induce an immune response by themselves and are immunogenic only after binding with the carrier. Polypeptides are common haptens.

The advantages of drugs synthesized from polypeptides are: (1) strong specificity; (2) Low toxic side effects; (3) High pharmacological activity; (4) The required dose is small; Disadvantages: (1) The purification process is complex; (2) High cost; (3) It is difficult to mass produce; (4) Poor metabolic stability (5) short half-life.

Polypeptide is widely used in medicine, beauty, the healthcare industry, and other fields, with more and more medical and health problems, the demand for polypeptide products is increasing. In beauty and health care, it is used daily as a skin care product for aging. In medicine, peptides can be used to make vaccines, and drugs.

Principle

Polypeptide is an intermediate product of protein hydrolysis and a compound formed by peptide bonds connecting amino acids. We are named in this way, and the compounds formed by dehydration and condensation of n amino acid molecules are called N-peptides, more than three peptides are called polypeptides.

Fig 1. Polypeptide chain diagram of EGS/NADP monomer orthogonal diagram

Synthesis method

(1) Chemical synthesis

①Liquid phase synthesis: including stepwise synthesis and fragment combination. Advantages of step-by-step synthesis: the synthesis step is simple and the experiment is completed quickly; Fragment combination: This method is more suitable for polypeptides containing more than 100 amino acids.

②Combinatorial chemistry: Amino acids are connected by combination, and compounds (physical and chemical properties or pharmacological activity) are screened from the obtained library, and applied to the discovery of drug leads.

(2) Biosynthesis

①Enzymatic method: The use of biological enzymes to degrade animal and plant proteins, to obtain peptides. Advantages: The nutritional value of protein is retained by this method; Disadvantages: This method has low yield, a long experiment period, and serious environmental pollution.

② Fermentation method: The use of microbial metabolism to obtain peptides. Advantages: It costs very little money; Disadvantages: This method is limited to cyanobacteria peptides, ε-polylysine, and γ-polylglutamate.

Hapten coupling

① Coupling background

Peptides as immunogens can‘t produce an immune response after immunizing the animal that belongs to non-immunogenic haptens, so it is necessary to conjugate small molecule hapten with a large molecular carrier protein (hapten coupling) to prepare antibodies.

Peptide coupling drugs are formed by the covalent combination of polypeptides and cytotoxic drugs through linkers, and many drugs are used in the targeted treatment of diseases. It has the advantages of circulation stability, and targeting, and can reduce the toxic side effects. In addition, structurally altered peptides make it easier for conjugated drugs to pass through various barriers, such as the blood-brain barrier.

Overall, Peptide coupling drugs represent a new direction in drug research and development, promising safer and more effective options for treating diseases.

② Coupled carrier:

The commonly used protein carriers mainly include bovine serum albumin (BSA), rabbit serum albumin (RSA), human serum albumin (HSA), oval albumin (OVA), hemocyanin (KLH), etc.

③ Coupling mode

Haptens are usually linked to carriers by physical and chemical methods.

It is difficult for small molecule antibodies to be directly coupled to the carrier or conformational changes will be caused after the connection, which are connected to the protein large molecule carrier through the connecting arm and Liker.

It is easy for polypeptides to be coupled to the carrier. The carboxylic or amino group on the polypeptide chain is linked by EDC (carbodiimide) or EDC and NHS(n-hydroxysuccinimide). A more efficient way is to add a Cys to the polypeptide and to be oriented to the protein carrier by Sulfo-SMCC reagent.

Conjugation of synthetic antigens to the carrier is generally 10-20 conjugation on a carrier, which is also an enrichment process for antigen epitopes. Synthetic peptides are mainly used for those antigens that are difficult to extract from the solution and achieve through recombinant expression or to detect a specific protein that is itself highly homologous to other proteins. The synthesis of polypeptides is a mature technology in the modern chemical industry. The main difficulty in selecting antigens is the epitope.

Based on the above content, KMD Bioscience has a relatively mature peptide synthesis platform, which can provide peptide synthesis, peptide coupling, Small molecule antigen production, antibody production, and other related experimental services. In addition, KMD Bioscience can provide services such as the common polypeptide antibodies, the preparation of antibody production, and the production of customized antibodies.

References:

[1] Kim SY, Hakoshima T. GST Pull-Down Assay to Measure Complex Formations [J]. Methods Mol Biol. 2019, 1893: 273-280.

[2] Lyu S, Zhang C, Hou X, Wang A. Tag-Based Pull-Down Assay [J]. Methods Mol Biol. 2022, 2400: 105-114.

[3] Louche A, Salcedo SP, Bigot S. Protein-Protein Interactions: Pull-Down Assays [J]. Methods Mol Biol. 2017, 1615: 247-255.