Protein-Protein Conjugation Methods
Protein-protein conjugation is commonly used in immunoassays for enzyme-labeled antibodies or antigens, essentially preparing signal markers. The main protein-protein conjugation methods include the periodate oxidation method (direct conjugation), the bis-maleimide method (homobifunctional conjugation), and the thiol-maleimide method (heterobifunctional conjugation).
1. Periodate Oxidation Method
The periodate oxidation method is a direct conjugation approach, primarily used when at least one of the target proteins is a glycoprotein. Periodate oxidizes glycosyl groups, converting vicinal diols into aldehyde groups. These aldehydes react with amino groups on another protein via a Schiff base reaction, forming a conjugate. The reaction product can be further reduced using sodium borohydride to form a more stable secondary amine (-NH-) structure. This method is mainly used for preparing HRP conjugates and labeling the Fc region of IgG.
2. Bis-Maleimide Method
The bis-maleimide method utilizes a homobifunctional reagent containing two maleimide groups to link thiol groups. When both target proteins contain small amounts of thiol groups, this method can be employed. Among such reagents, N,N’-o-phenylenedimaleimide (PDM), which has dual thiol-reactive maleimide groups, is the most widely used. This method effectively prevents the formation of protein aggregates or cross-linking caused by glutaraldehyde reacting with protein amino groups. In immunoassays, this method is particularly suitable for conjugating β-galactosidase with antibody fragments such as Fab or Fab’.
3. Thiol-Maleimide Method
The thiol-maleimide method is a heterobifunctional conjugation technique that allows better control over product composition. This method typically requires two reagents: one containing an amine-reactive group along with a thiol, and another containing an amine-reactive group along with a maleimide. Free thiols tend to spontaneously form undesired dimers; therefore, in practical applications, thiols are often used in a protected form, such as S-acetyl derivatives.
Among protein-protein conjugation methods, heterobifunctional conjugation based on thiol-maleimide reactions is predominant. Additionally, maleimide can be replaced by other reactive groups for thiol conjugation, such as the disulfide-containing compound SPDP.
Protein-Small Molecule Conjugation Methods
Protein-small molecule conjugation has two important applications in immunoassays. First, it is used for biotin labeling of enzymes or antibodies, facilitating protein-protein conjugation through biotin-streptavidin bridging while minimizing conformational changes in protein molecules. Second, it enables the labeling of antibodies or antigens with fluorescent or luminescent groups to generate signal markers. Protein-small molecule conjugation methods are typically based on carboxyl-amino reactions. Additionally, Mannich reactions or commercially available ready-to-use conjugation reagents offer alternative solutions. The main protein-small molecule conjugation methods include the carbodiimide method, carbodiimide/N-hydroxysuccinimide (NHS) ester method, mixed anhydride method, Mannich reaction, and the use of commercially available ready-to-use conjugation reagents.
1. Carbodiimide Method and Carbodiimide/NHS Ester Method
Carbodiimides activate carboxyl groups to form O-acylisourea intermediates, which then react with protein amino groups for conjugation. Carbodiimides are classified into water-soluble and non-water-soluble types. Dicyclohexylcarbodiimide (DCC) is the most common non-water-soluble carbodiimide, while the widely used water-soluble carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC).
A typical DCC conjugation reaction follows a two-step process: first, DCC is introduced in a water-miscible organic solvent to derivatize the small molecule, and then the resulting product is added to a protein aqueous solution for conjugation. When using EDAC for carboxyl activation, activation and conjugation can occur in a single step. However, a drawback is that the optimal pH for isourea formation is 5-6, which is significantly lower than the optimal pH range (9-10) for amino group activity. Therefore, even when using EDAC for carboxyl activation, the reaction is often performed in separate steps to improve yield.
In carbodiimide conjugation, the isourea intermediate is often converted in situ into a succinimide ester, which then reacts with the protein’s amino groups for conjugation, effectively increasing the yield. Typically, a mixture of N-hydroxysuccinimide (NHS) and DCC is used to activate carboxyl groups in an organic solvent before being added to the protein aqueous solution. Water-soluble N-hydroxysulfosuccinimide (NHSS) can be used in the one-step EDAC method. In terms of conjugation efficiency, the two-step DCC/NHS synthesis method offers a higher yield and is the preferred choice for conjugating carboxyl-containing compounds with proteins. Pure aqueous-phase environments can prevent organic solvents from affecting proteins, but the yield is impacted by hydrolysis.
2. Mixed Anhydride Method
Anhydrides are compounds formed by the dehydration of two carboxyl groups and can remain stable in aqueous solutions while reacting with protein amino groups. In practical applications, a mixed anhydride intermediate is often prepared by reacting chloroformates with small molecules in an anhydrous environment, followed by conjugation with proteins in an aqueous phase. Isobutyl chloroformate is the most commonly used chloroformate reagent.
3. Mannich Reaction
The Mannich reaction typically has a lower reaction rate and yield but serves as an alternative when conventional conjugation methods are challenging to implement. In this reaction, protein amino groups react with carbon atoms from various organic compounds in the presence of an aldehyde (commonly formaldehyde). The carbon atom must contain an active hydrogen atom that is readily displaced.
4. Ready-to-Use Conjugation Reagents
With the growing demand for protein modification and antibody conjugation, many ready-to-use conjugation reagents are now commercially available. Biotin-NHS (biotin N-hydroxysuccinimide ester) is a widely used commercial biotinylation reagent that conjugates with protein amino groups. Fluorescent or luminescent group derivatives, available in NHS ester or isothiocyanate forms, can also be obtained commercially. These ready-to-use reagents offer a convenient and effective method for protein conjugation, ensuring high yield while preserving protein activity.
The resulting protein-protein or protein-small molecule conjugates are usually mixtures containing the target product, unreacted proteins, or small molecule monomers. In practical applications, further purification and characterization of the obtained mixtures are necessary.
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