New drug discovery: Protein-protein interactions

Protein-protein interactions (PPI) are highly specific electrostatic attractions between protein structures. The interactions regulate cell function and influence physiology and development. Mass spectrometry is often used to detect protein-protein interactions. However, all proteomic screens are differential and require two samples such as IP and mock IP, POI and wildtype, or POI and knockout.

The peptide/ biotin-peptide or peptide/fluorescence-labeled peptide are excellent tools for studying protein-protein interactions. Check this link for details: Protein-Protein Interactions: Methods for Detection and Analysis.

The compounds that can modulate PPI are hard to discover because the proteins have multiple binding sites and the screening assays are not reliable. In many cases, two or more proteins may interact with one another and form a complex. The optical fluorescence-based methods such as the Cy5, Cy7, FAM, FITC, TAMRA-labeled peptides, or FRET assay are particularly useful in these circumstances. Click for more details:

The interactions between a fluorescently labeled or intrinsically fluorescent sample and a binding patterner are measured during the application. The changes in intrinsic fluorescence from tryptophan and tyrosine residues in the protein can be measured, which indicates transitions in the protein’s folding state.

The scientists have been working on fusion-based bifunctional proteins in cancer immunotherapy. The bifunctional protein sent an apoptotic signal to the tumor cells and enhanced their killing. The click chemistry is the perfect tool for the drug-protein or protein-protein conjugation. The more we understand the natural receptor-ligand complex and how it might signal, the better we can guide the design of therapeutic agonists. Click here for the peptide conjugation details:

Simple method to prepare antibody-peptide, antibody-oligonucleotide or antibody-compound conjugates

We describe a simple method for preparing antibody-peptide, antibody-oligonucleotide or antibody-compound conjugates and discuss its applications in drug delivery and new drug design. Conjugation is based on alkyne-azide cycloaddition. This Cu-free click reaction starts from the dibenzocyclooctyne (DBCO) moiety-activated antibodies and subsequently linked covalently with an azide-modified peptide, oligonucleotide or compounds. The reaction is performed under physiological conditions and has no adverse effects on antibodies or proteins. This can also be used as the click chemistry fluorescence labeling and the click chemistry in peptide-based drug design.

However, the copper-catalyzed alkyne-azide cycloaddition (CuAAC) is not suitable for applications involving functional biomolecules because copper ions can cause protein denaturation.

Measuring the protein levels directly is challenging. However, the signals can be amplified by immuno-PCR using oligonucleotide-attached antibodies to detect protein indirectly.




Preparing Antibody-Peptide, Antibody Oligonucleotide or Antibody-Compound Conjugates

1. Conjugation of DBCO to the Antibody. The DBCO-PEG5-NHS was used to react with the NH2 groups on the antibody. The inclusion of a PEG5 linker improves the water solubility of the hydrophobic DBCO, introduces a spacer and flexibility between the antibody molecule and the peptide/oligonucleotide or compounds. This will alleviate the steric effect of the antibody on the enzymatic reactions.

2. Prepare the azido-Peptide or azido-oligonucleotide. LifeTein provides click chemistry modified peptide synthesis: N-terminal azide-peptide/oligo or C-terminal peptide/oligo-azide.

3. Covalent attachment of the peptide/oligonucleotide to the antibody. The reaction between DBCO and azide is slow compared to CuAAC reaction. The reaction time of 16–18 h in PBS at 4 °C is ideal to increase the final product yield. The DBCO-antibody in the intermediate reaction is stable.

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Simple method to prepare antibody-peptide, antibody-oligonucleotide or antibody-compound conjugates