Related Experiment Video

Updated: Aug 14, 2026

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling
08:51

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

Published on: June 25, 2015

Gi/o proteins: expression for direct activation enquiry

Lorenzo Di Cesare Mannelli1, Alessandra Pacini, Annarita Toscano

  • 1Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50134 Florence, Italy. lorenzo.mannelli@unifi.it

Protein Expression and Purification
|December 21, 2005
PubMed

Insights

Researchers expressed and characterized human G protein subunits for evaluating new compound activation profiles. These recombinant subunits are stable, folded, and active, suitable for functional studies in cell signaling.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Signaling

Background:

  • G protein-mediated pathways are crucial for cellular communication.
  • Understanding G protein subunit function is key to developing targeted therapeutics.
  • Characterization of specific G protein subunits (alphai1, alphai3, alphao1, beta1, gamma2) is essential for detailed pathway analysis.

Purpose of the Study:

  • To describe the expression and characterization of key human G protein subunits.
  • To develop a robust method for evaluating G protein activation by novel compounds.
  • To assess the functionality of recombinant G protein subunits.

Main Methods:

  • Engineered pCR-TOPO T7 vectors for target G protein subunit gene insertion.
  • Over-expressed subunits in Escherichia coli as His-tagged fusion proteins.
  • Purified subunits using metal chelate chromatography and removed His-tags via enterokinase digestion.
  • Analyzed secondary structures using circular dichroism.
  • Assessed functionality via GTP hydrolysis and GTPgammaS binding assays in the presence/absence of modulators (Mastoparan, ML250).

Main Results:

  • Successfully expressed and purified recombinant human G protein subunits (alphai1, alphai3, alphao1, beta1, gamma2).
  • Confirmed proper folding and stability of recombinant subunits through circular dichroism.
  • Demonstrated full functionality of subunits in GTP hydrolysis and GTPgammaS binding assays.
  • Showcased the utility of these subunits in assessing modulators like Mastoparan and ML250.

Conclusions:

  • Recombinant human G protein subunits are stable, correctly folded, and fully active.
  • These characterized subunits are suitable for functional studies and evaluating new compound effects on G protein activation.
  • The developed approach provides a valuable tool for drug discovery in G protein-mediated signaling pathways.

More Related Videos

Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters09:22

Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters

Published on: November 26, 2013

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay11:12

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

Published on: February 22, 2018

Related Concept Videos

RNA Polymerase II Accessory Proteins02:36

RNA Polymerase II Accessory Proteins

Proteins that regulate transcription can do so either via direct contact with RNA Polymerase or through indirect interactions facilitated by adaptors, mediators, histone-modifying proteins, and nucleosome remodelers. Direct interactions to activate transcription is seen in bacteria as well as in some eukaryotic genes. In these cases, upstream activation sequences are adjacent to the promoters, and the activator proteins interact directly with the transcriptional machinery. For example, in...
Cell Specific Gene Expression01:58

Cell Specific Gene Expression

Multicellular organisms contain a variety of structurally and functionally distinct cell types, but the DNA in all the cells originated from the same parent cells. The differences in the cells can be attributed to the differential gene expression. Liver cells, whose functions include detoxification of blood, production of bile to metabolize fats, and synthesis of proteins essential for metabolism, must express a specific set of genes to perform their functions. Gene expression also varies with...
Cell Specific Gene Expression01:58

Cell Specific Gene Expression

Multicellular organisms contain a variety of structurally and functionally distinct cell types, but the DNA in all the cells originated from the same parent cells. The differences in the cells can be attributed to the differential gene expression. Liver cells, whose functions include detoxification of blood, production of bile to metabolize fats, and synthesis of proteins essential for metabolism, must express a specific set of genes to perform their functions. Gene expression also varies with...
Prokaryotic Transcriptional Activators and Repressors01:58

Prokaryotic Transcriptional Activators and Repressors

The organization of prokaryotic genes in their genome is notably different from that of eukaryotes. Prokaryotic genes are organized, such that the genes for proteins involved in the same biochemical process or function are located together in groups. This group of genes, along with their regulatory elements, are collectively known as an operon. The functional genes in an operon are transcribed together to give a single strand of mRNA known as polycistronic mRNA.
Transcription of prokaryotic...
Prokaryotic Transcriptional Activators and Repressors01:58

Prokaryotic Transcriptional Activators and Repressors

The organization of prokaryotic genes in their genome is notably different from that of eukaryotes. Prokaryotic genes are organized, such that the genes for proteins involved in the same biochemical process or function are located together in groups. This group of genes, along with their regulatory elements, are collectively known as an operon. The functional genes in an operon are transcribed together to give a single strand of mRNA known as polycistronic mRNA.
Transcription of prokaryotic...
Reporter Genes02:11

Reporter Genes

Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
Commonly used reporter...