Protein-Peptide Conjugation

With a comprehensive state-of-the-art platform, BOC Sciences is fully capable and committed to providing one-stop molecular bioconjugation services. Peptides alone are often too small to produce an antigenic immune response. Therefore, the production of anti-peptide antibodies needs to be coupled with large carrier proteins. When administered in the body, the immune system reacts to these peptide-protein conjugates to produce antibodies. As a leading service provider in the field of drug discovery and research, BOC Sciences provides global clients with protein-peptide conjugation services to promote your research.

What is protein-peptide conjugation?

Protein-peptide conjugation is a process where a peptide (a short chain of amino acids) is covalently attached to a protein molecule. The carrier protein can be conjugated with a peptide on the N-terminal, C-terminal or internal residue. This conjugation can be achieved through various chemical methods and serves several purposes, including enhancing the stability, bioactivity, or targeting capabilities of the protein. This technique holds immense promise in drug development, diagnostics, and biotechnology due to its ability to combine the advantageous properties of both proteins and peptides.

Purpose of protein-peptide conjugation

Stability: Peptide conjugation can improve the stability of a protein against degradation by proteases or other enzymes.

Bioactivity: By attaching specific peptides, such as cell-penetrating peptides or targeting sequences, the bioactivity of the protein can be modified or enhanced.

Targeting: Peptides can serve as targeting moieties to direct the protein to specific cells or tissues, improving its therapeutic efficacy.

Solubility: Conjugation with peptides can enhance the solubility of proteins, which can be beneficial for formulation and delivery.

Protein-peptide conjugation services

BOC Sciences, a leading service provider in the field of drug discovery and research, can provide protein peptide binding services to clients worldwide to facilitate your research. We can perform Protein-peptide conjugation using a variety of methods, each with its advantages and suitability depending on the specific application.

Chemical crosslinking

NHS ester coupling: This method involves using N-hydroxysuccinimide (NHS) esters to react with primary amines (lysine residues) on the protein and the N-terminus of the peptide. This forms a stable amide bond.

Maleimide chemistry: Maleimide groups can react specifically with thiol groups (-SH) on cysteine residues of the protein, providing a stable linkage.

Enzymatic ligation

Transpeptidation: Enzymes like sortase or microbial transglutaminase can be used to catalyze the ligation between a specific peptide sequence (containing a recognition motif) and a protein substrate. This results in a site-specific and controlled conjugation.

Photocrosslinking

We also offer a photocrosslinking method for protein-peptide crosslinking. Photocrosslinking utilizes photosensitive molecules that are activated by light at specific wavelengths to produce reaction intermediates capable of forming covalent bonds with nearby proteins or peptides.

Native chemical ligation (NCL)

NCL involves the use of a thioester peptide and a cysteine-containing peptide. The thioester reacts with the cysteine sulfhydryl group to form a native peptide bond. This method is effective for complex conjugations.

Biotin-streptavidin linkage

Biotin can be attached to the peptide, and streptavidin, a tetrameric protein with high affinity for biotin, can then bind to the biotinylated peptide. This linkage is strong and can be used in various applications, including immobilization.

Click chemistry

Reactions like copper-catalyzed azide-alkyne cycloaddition (CuAAC) or strain-promoted azide-alkyne cycloaddition (SPAAC) can be utilized for conjugation. Peptides or proteins are modified with azide or alkyne groups and react selectively with complementary functional groups.

Genetic fusion

In genetic engineering, genes encoding a peptide of interest can be fused to a gene encoding a protein. The resulting recombinant protein expresses the peptide as part of its structure.

Hybrid methods

Combinations of chemical and enzymatic methods can offer site-specific and controlled conjugation. For instance, combining NHS ester chemistry with enzymatic ligation for dual-site conjugation.

Optimization of protein-peptide conjugation

Selection of coupling chemistry

Choose an appropriate chemistry based on the functional groups present on both the protein and peptide. Consider the stability of the conjugation chemistry under physiological conditions.

Protein and peptide purification

Ensure both the protein and peptide are purified to remove contaminants that could interfere with conjugation. Use techniques such as size exclusion chromatography (SEC) or high-performance liquid chromatography (HPLC) to purify the protein and peptide.

Optimizing reaction conditions

Determine the optimal pH and buffer conditions for the coupling reaction. The stability and solubility of both the protein and peptide should be considered. Test different temperatures and incubation times to optimize the reaction efficiency without compromising protein stability.

Molar ratios

Determine the optimal molar ratio of protein to peptide for conjugation. This ratio can affect the extent of conjugation and the homogeneity of the final product. Use excess peptide to drive the reaction towards completion but avoid using excessively high ratios that may lead to nonspecific conjugation or aggregation.

Monitoring conjugation

Use analytical techniques such as SDS-PAGE, mass spectrometry, or HPLC to monitor the progress of the conjugation reaction. Assess the degree of conjugation and identify any side products or aggregates.

Purification of conjugated product

After conjugation, purify the protein-peptide conjugate to remove unconjugated protein, peptide, and any side products. Again, use techniques such as SEC or HPLC to isolate the desired conjugated product.

Validation of protein-peptide conjugation

Gel electrophoresis analysis: Gel electrophoresis can be used to assess the effectiveness of a cross-linking reaction. In gel electrophoresis, cross-linked proteins or peptides typically show higher molecular weights and form new bands or bands.

Mass spectrometry: Mass spectrometry allows the identification and characterization of the conjugate and the location of cross-linking sites.

Western blotting: By immunoblotting cross-linked proteins or peptides, the presence of protein-peptide conjugate can be detected and their relative abundance determined.

Binding assays: Perform binding assays (e.g., ELISA, surface plasmon resonance) to assess whether the conjugate retains the ability to interact with its target.

Functional evaluation: Conduct functional assays relevant to the protein's activity (e.g., enzymatic assays, cell-based assays) to ensure that the conjugation does not impair functionality.

Examples of frequently used carrier proteins for peptide conjugates

• KLH (keyhole cap hemocyanin) is the most commonly used carrier protein, which has higher immunogenicity compared with other proteins. KLH is commonly used in vertebrate research.
• BSA (Bovine serum albumin) is a commonly used weak antigenic compound carrier protein because it contains a large number of binding lysine residues.
• HSA (Human serum albumin).
• OVA (Ovalbumin) is a protein isolated from eggs. It is often used as a control carrier protein to verify whether the antibodies produced by KLH/BSA are specific to target peptide and carrier proteins.

Why choose BOC Sciences?

• Comprehensive and proficient bioconjugation technical platform
• All-round cross-linking chemistry
• Multiple methods for protein peptide binding validation
• Data analysis, detailed report with results and discussion
• Partner-like relationship
• Industry-experienced personnel
• Designated project management team
• 24h-available customer service

FAQ

1. What are the applications of protein-peptide conjugation?

Protein-peptide conjugation is used in various biomedical applications including targeted drug delivery, immunotherapy, vaccine development, and diagnostic assays.

2. How is protein-peptide conjugation performed?

Protein-peptide conjugation is typically achieved through chemical or enzymatic methods. Common approaches include amine-reactive crosslinkers, thiol-reactive crosslinkers, and specific enzyme-mediated conjugation.

3. What types of peptides can be conjugated to proteins?

A wide range of peptides can be conjugated, including linear peptides, cyclic peptides, cell-penetrating peptides, and peptide mimetics.

4. What factors should be considered when designing a protein-peptide conjugation experiment?

Important considerations include the choice of conjugation chemistry, site-specific versus random conjugation, preservation of protein function, stability of the conjugate, and scalability for production.

5. How can protein-peptide conjugation enhance protein function?

Conjugating a peptide can modulate protein stability, solubility, targeting specificity, or immunogenicity, depending on the desired application.

6. What analytical techniques are used to characterize protein-peptide conjugates?

Characterization methods include mass spectrometry, chromatography (HPLC, SEC), SDS-PAGE, Western blotting, and bioactivity assays.

7. What scale of protein-peptide conjugation services do you offer?

We offer protein-peptide conjugation services ranging from small-scale research quantities to large-scale production batches, tailored to meet your specific needs.

8. What is the typical turnaround time for protein-peptide conjugation projects?

Turnaround times can vary depending on the scope of the project and specific requirements. Our team will provide you with an estimated timeline based on your project details.