PharmiWeb.com - Global Pharma News & Resources
01-Jul-2022

GST-Pull Down Technology

Summary

The functioning of biological systems is inextricably linked to protein-protein interactions, and important biological processes such as DNA synthesis, gene transcriptional activation, protein translation, modification and localization, and information transfer all involve the role of protein complexes.
  • Author Name: Melissa George
Editor: Melissa George Last Updated: 05-Jul-2022

Introduction to protein interactions

The functioning of biological systems is inextricably linked to protein-protein interactions, and important biological processes such as DNA synthesis, gene transcriptional activation, protein translation, modification and localization, and information transfer all involve the role of protein complexes. Therefore, it is particularly important to discover and verify protein-nucleic acid and protein-protein interactions in living organisms. Various techniques have been developed for studying protein-protein interactions, such as pull down technique, yeast two-hybrid (Y2H) technique, immunoprecipitation technique, phage display technique, surface plasmon resonance (SPR) technique, fluorescence resonance energy transfer (FRET), isothermal titration calorimetry (ITC), cell co-localization technique, etc.

 

Pull-down techniques are commonly used for in vitro protein interaction assays, in combination with LC-MS/MS techniques to discover new interacting proteins. The basic principle is to use a solid-phase, labeled decoy or tag protein (biotin-, PolyHis-, or GST-) to fish for an unknown target protein interacting with it from a target lysate. The decoy protein (tag protein) is immobilized on some substrate and co-incubated with the target lysate. Target proteins that can interact with the decoy protein are adsorbed by the immobilized substrate, while impurities that are not adsorbed are eluted off. The target protein can then be obtained by changing the elution conditions. The pull-down technique allows the interaction between known and unknown proteins to be determined.

 

Convergence Labeling

Fusion tags are specific protein, peptide or oligopeptide tags expressed by fusion at the N-terminal or C-terminal end of the target protein using DNA in vitro recombination techniques. Recombinant proteins are bound by fusion tags to specific ligands encapsulated on a solid phase matrix, allowing for targeted immobilization and purification of recombinant proteins, greatly simplifying the detection of recombinant proteins. It retains most of the structure of natural proteins, but also increases solubility, prevents degradation, promotes secretion, and facilitates purification.

 

GST - pull down technology

The GST-pull down technique was developed on the basis of GST (glutathione-S-transferase) fusion proteins. Since Smith and his team purified GST-tagged fusion proteins in 1988, it has gradually become a major tool for studying protein-protein interactions in vitro. the GST-pull down principle consists of the following main components:

  • Obtaining fusion proteins with GST tags.
  • Affinity of the GST-tagged fusion protein onto the glutathione chromatography column.
  • Acquisition of proteins that may interact with the fusion protein.
  • Interacting proteins are affixed to the glutathione chromatography column.
  • Cut off the tag and elute to obtain the interacting proteins.

 

GST is fused to the tagged protein and the fused protein can be expressed in many expression systems.

 

Application of GST-pull down technology

GST-pull down technique is mainly used to study strong or stable protein interactions in vitro, which can verify the possible direct interaction of two known proteins or find unknown target proteins that may interact with the target protein, and has strong specificity when verifying direct protein interactions in vitro.

 

Creative Proteomics has extensive experience in proteomics services. In order to better study molecular interactions, a new division has been set up specifically for protein interaction experiments to optimize the technology to overcome technical difficulties. Creative Proteomics has launched protein pull-down service, which can be customized to meet project needs.