Peptide synthesis and antigen preparation
Peptide synthesis and antigen preparation
Hapten is a substance that is only immune reactive, but not immunogenic, so it is also called incomplete antigen. Small molecules belong to haptens, which need to be connected with large molecular substances (carriers) to form immunogenicity, which can induce an immune response and produce antibodies. Chemically synthesized peptides, which are difficult to have good antigenicity, can only induce a very weak immune response in animals, and therefore need to be cross-linked with carrier proteins[1].
1. Selection of polypeptide antigens
The selected polypeptide sequence is usually 15-20 bases, a single epitope usually contains 5-8 bases, and a 15-20 peptide usually contains 1 or more epitopes. Longer peptides are better able to maintain consistency with natural proteins and are more likely to produce antibodies. Since peptides are synthesized by chemical methods, we also need to take into account the difficulty of peptide synthesis and its good solubility.
We should minimize the selection of peptides containing hydrophobic bases (such as tryptophan, isoleucine, valine, etc.). Glutamine is easy to form hydrogen bonds with peptide chains, resulting in insoluble peptides, so polypeptides with multiple glutamates should also be avoided.
Cysteine facilitates the coupling of polypeptides to carrier proteins. Therefore, the N-terminal or C-terminal cysteine of polypeptide should be retained in order to facilitate carrier protein coupling and thus have better immunogenicity. The presence of two or more cysteines at the same time needs to be avoided because it can cause disulfide bonds to form between polypeptide chains, leading to insolubility and structural changes. When the chosen polypeptide lacks cysteine, we can add cysteine to the n-terminal or C-terminal.
2. Carrier protein[2]
Carrier proteins have many antigenic determinant groups, commonly used are keyhole limpet hemacyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA).
KLH | BSA | OVA |
Hemocyanin is a free blue respiratory pigment found in the hemolymph of certain mollusks and arthropods (spiders and beetles). Hemocyanin contains two copper ions directly connected to the polypeptide chain, similar to iron-containing hemoglobin, it is easy to bind to oxygen, but also easy to dissociate with oxygen, is known to be the only reversible binding with oxygen copper protein, oxidation is blue green, reduction is white. Its molecular weight is 450,000 ~ 130,000. Because KLH has a higher immunogenicity than BSA, it is the most commonly selected carrier protein | Bovine serum albumin (BSA) is a plasma protein derived from cattle with a molecular weight of 7×104Da (containing 59 Lys), which is among the most stable and soluble albumin. About 30-35 major amino groups are available for conjugation with linkers, making BSA a popular carrier protein for weak antigenic compounds. Because BSA is smaller than KLH, it is more soluble in water than KLH. | Ovalbumin (OVA) is the most abundant protein in egg white, with a molecular weight of 4.5×104Da. It is often used as a second carrier protein to confirm that the antibody is a specific peptide and not a carrier protein, such as bovine serum albumin. |
3. Coupling method[3]
A sulfhydryl modification (via the side chain of Cys) is used to conjugate KLH, BSA or OVA, and all functional groups carrying the sulfhydryl reaction can be modified to conjugate the reaction.The most common are the following:
3.1 A coupling containing carboxyl groups or carboxylable haptens：
Mixed anhydride method | Carbodiimide |
Mixed anhydride method, also known as isobutyl chloroformate method. The carboxyl group on hapten reacts with isobutyl chloroformate in the presence of n-butylamine to form an intermediate of mixed acid anhydride, and reacts with amino group of protein to form hapten-protein conjugate. | Carbodiimide (EDC) dehydrates the carboxyl group and the amino group to form an amide bond. The carboxyl group on the hapten reacts with the EDC to form an intermediate, and then reacts with the amino group on the protein to form the hapten-protein conjugate. EDC is known as one of the zero-length crosslinkers because it does not form arm molecules as a medium for the formation of amide bonds. This method of connection is very simple, just the carrier protein and antigen mixed in a certain proportion of the appropriate solution, then add water-soluble carbonized diimide, stir 1-2h, room temperature 24h, and then dialysis can be. If the hapten molecule does not contain carboxyl groups, carboxyl groups can be introduced through certain chemical reactions. After the introduction of carboxyl groups, the coupling can also be performed by the above method. |
3.2 Coupling of haptens containing an amino or reducible nitro group：
Glutaraldehyde process | Diazotization |
The two aldehyde groups of the bifunctional agent glutaraldehyde form schiff bonds (-N=C <) with the hapten and the amino group on the protein, respectively, introducing a five-carbon bridge between the hapten and the protein. This reaction condition is mild, can be carried out in 4-40℃ and pH6.0-8.0, the operation is also simple, so it is widely used. Glutaraldehyde is affected by light, temperature and alkalinity, and may self-polymerize, weakening its cross-linking effect, so it is best to use fresh glutaraldehyde. | The active group is the hapten of the aromatic amine group, which reacts with NaNO2 and HCl to obtain a diazo salt, which can be directly attached to the ortho-site of the carboxyl group of the protein tyrosine to form an azo compound. |
3.3 Conjugation of hydroxy-containing haptens：
Succinic anhydride method | Carbonyl diimidazole method |
The hapten hydroxyl group reacts with succinic anhydride in anhydrous pyridine to obtain a half-succinate (an intermediate with carboxylic group), which is then combined with a protein amino group by carbodiimide or mixed anhydride method, and a succinate group is inserted between the hapten and the protein carrier. | N, N-carbonyl diimidazole is a highly active reagent for the introduction of carbonyl groups and was first shown to be an excellent amide bonding reagent in peptide synthesis. The carboxyl-containing molecule reacts with the carbonyl diimidazole to form an intermediate imidazole formate, which can react with N-nucleophiles to obtain n-alkylated formate bonds. Generally, proteins form uncharged urane-like derivatives through the n-terminal (α-amino) and lysine side chain (e-amino) and molecules, which have excellent chemical stability. |
KMD Bioscience (https://www.kmdbioscience.cn/) provides standard linear peptides, and can provide a variety of modified peptide synthesis services according to customer's project requirements. Modified peptides are widely used in peptide drug research, immunology, diagnostics, biocatalysts, antibody modification and peptide reagents. KMD Bioscience provides a variety of peptide modification services, we can amidate or acetyl peptide sequences, modify disulfide bonds, and also label peptides; We mainly use BSA, KLH and OVA for peptide coupling services.
The peptide modification and coupling services provided by KMD Bioscience are based on the extensive experience of our technical experts and advanced technologies, and we can meet the various needs of our customers.
This article serves as a reference material for enthusiasts in scientific research. It does not substitute for professional knowledge or hands on experimental procedures which require more detailed and professional information. In case of any content infringement, kindly reach out to the author for immediate deletion of the contentious material.
[1] Geysen H M .Antigen-antibody interactions at the molecular level: adventures in peptide synthesis[J].Immunology Today, 1985, 6(12):364-369.DOI:10.1016/0167-5699(85)90096-9.
[2] Merrifield R B , Stewart J M .Automated Peptide Synthesis[J].Nature[2023-10-26].DOI:10.1038/207522a0.
[3] Benoiton N L .Chemistry of Peptide Synthesis[J].british journal of venereal diseases, 2006.DOI:10.1136/sti.60.2.117.
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