Monthly Archives: August 2018

New drug discovery: Protein-protein interactions

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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.
https://www.lifetein.com/Peptide_Modifications_biotinylation.html

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: https://www.lifetein.com/Peptide-Synthesis-FITC-modification.html.

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: https://www.lifetein.com/price_modification_labeling.html

A Versatile Approach: Epitope Mapping Simplified with Synthetic Peptides

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Epitope mapping identifies antigen regions that serve as binding sites for antibodies. The overlapping linear peptides derived from the primary sequence of the antigen are frequently used for epitope screening. Individual peptides can be divided into several fragments that overlap. The resulting overlapping peptide libraries can then be used for processes including continuous and linear epitope mapping.

For example, to map the epitope of an antibody, a few overlapping fragments spanning the target regions are constructed in an expression vector. These constructs are transiently transfected in cells and whole cell lysates collected after 48 hours are subjected to Western blotting with the antibody. To further map the region of this fragment, a series of overlapping peptides are synthesized. These peptides and lysates from the cells expressing the target full-length gene or empty vector (negative) are performed by the dot blot.

Mapping epitopes quickly and accurately are challenging because the epitopes tend to be nonlinear on antigens. Combining binding specifically toward two distinct epitopes into a single molecule can significantly enhance the immunotherapeutic properties of monoclonal antibodies. Multivalent interactions are the most efficient at driving IgE receptor signaling pathways.

There are several useful tools for studying antigen-antibody interactions.

  1. Use the MAPs as the immunogens. Multiple Antigenic Peptides (MAPs) are peptides that are branched artificially, in which Lys residues are used as the scaffolding core to support the formation of 8 branches with varying or the same peptide sequences. For example, one goal is to include different epitopes from different virus proteins in a single unit. The epitopes showing the right prediction of antigenicity and conserved in most serotypes of a virus are selected and assembled as the MAP.
  2. Screening combinatorial peptide libraries to optimize enzyme substrates and create high-affinity protein ligands. A critical biological application of custom peptide libraries is the characterization of the binding events that occur between specific proteins and their peptide ligands. A series of Overlapping Peptide, Truncation Peptide, Alanine Peptide Scanning, Scrambled Peptide, or Positional Peptide can be used for mapping and validating epitopes, the characterization of therapeutic antibodies, studying anti-antibody and neutralizing antibody actions in vitro.
Synthetic Peptide Library

Cell Penetrating Peptide Examples and Peptide Synthesis

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Tat, the transcription activator of the human immunodeficiency virus type 1 (HIV-1) viral genome, enters cells in a non-toxic and highly efficient manner. Tat is the first known cell-penetrating peptide.

CPPs have been used as a carrier to deliver proteins or genes into cells and tissues. In this study, a CPP library composed of 55 CPPs was used to deliver genes into plant cells. Many CPPs showed efficient penetration into plant cells. The Lys-containing CPPs have higher penetration efficiency in the plant than in animal cells. This could be due to differences in lipid composition and surface charge of the cell membranes. No correlations were detected between the penetration efficiency and the cationic, amphipathic, or hydrophobic properties of peptides.

D-R9 is composed of D-form amino acids. D-R9 bound preferentially to the membrane and did not penetrate the cytosol or vacuole. In mammalian cells, poly-lysine-based CPPs are efficient and interact with membrane lipid head groups to induce wrapping of the membrane monolayers. Arg-rich peptides, such as the Tat peptide, are among the most efficient CPPs. Arg-rich CPPs may generate negative Gaussian membrane curvature to form pores or protrusions from endocytosis. The cell penetration efficiency of CPPs containing poly-Arg is higher than those containing poly-Lys. However, in a plant, Arg-rich CPPs are not the most efficient at penetrating plant cells.

CPP peptide property

CPP peptide property