Recombinant Human Guanine nucleotide-binding protein G (q) subunit alpha (GNAQ) is produced in an E.coli expression system, covering the complete 1-359 amino acid sequence. The protein carries a C-terminal 6xHis tag, which makes purification and detection straightforward. SDS-PAGE analysis shows the product reaches greater than 95% purity, likely making it appropriate for research applications that need high-quality protein.
GNAQ appears to play a critical role in intracellular signaling pathways. Being part of the G protein family, it participates in transducing extracellular signals that G protein-coupled receptors receive. GNAQ seems to regulate various physiological processes, including cell proliferation and survival, by interacting with downstream effectors. This importance in signaling pathways may make it valuable for research into cellular communication and signaling mechanisms.
Potential Applications
Note: The applications listed below are based on what we know about this protein's biological functions, published research, and experience from experts in the field. However, we haven't fully tested all of these applications ourselves yet. We'd recommend running some preliminary tests first to make sure they work for your specific research goals.
Based on the provided information, the recombinant human GNAQ protein is expressed in E. coli, a prokaryotic system that is generally unsuitable for producing functional eukaryotic G-protein alpha subunits. GNAQ requires precise folding, GTP-binding capability, and specific post-translational modifications (including lipid modifications) for its role in G-protein coupled receptor signaling. While the protein is full-length (1-359aa) with a C-terminal 6xHis tag and high purity (>95%), E. coli lacks the eukaryotic chaperones and modification machinery necessary for proper folding and prenylation of G-protein alpha subunits. The C-terminal His tag may particularly interfere with the critical C-terminal region that is essential for receptor interaction and membrane targeting. Since activity is unverified, the protein cannot be assumed to be correctly folded or bioactive without experimental validation of its GTP-binding and hydrolysis capabilities.
1. Protein-Protein Interaction Studies via His-Tag Pull-Down Assays
The C-terminal 6xHis tag enables technical feasibility for pull-down assays. However, if GNAQ is misfolded (as likely in E. coli), it will not interact physiologically with its true binding partners (e.g., G-protein coupled receptors, RGS proteins, effectors). G-proteins require precise conformational changes during GTP/GDP cycling for specific interactions. Identified interactions may be non-physiological artifacts. This application should not be pursued without confirmation of proper folding and GTP-binding activity.
2. Antibody Development and Validation
The recombinant GNAQ can serve as an effective immunogen for generating antibodies that recognize linear epitopes, even if misfolded. The full-length sequence ensures broad epitope coverage. However, antibodies may not recognize conformational or modification-dependent epitopes of native, lipid-modified GNAQ in cells. Validation against endogenous GNAQ from mammalian systems is essential.
3. Biochemical Characterization and Stability Studies
This application is well-suited for assessing the recombinant human GNAQ protein itself. Techniques like circular dichroism spectroscopy, size-exclusion chromatography, and thermal shift assays can evaluate the protein's folding state and stability. These studies are valuable even if the protein is inactive, as they characterize the recombinant human GNAQ protein and can inform about its suitability for other applications.
4. Competitive Binding Assays and Inhibitor Screening
This application is highly problematic without activity verification. If GNAQ is misfolded, it will not bind GTP/GDP properly or interact with regulators/effectors with correct specificity. Screening campaigns would identify compounds that target non-physiological conformations. This application requires prior validation of proper folding, GTP-binding, and hydrolysis activities.
Final Recommendation & Action Plan
Given the extreme challenges of producing functional G-protein alpha subunits in E. coli, we recommend first performing comprehensive biophysical and functional validation: 1) Biophysical characterization (circular dichroism for secondary structure, analytical ultracentrifugation for oligomeric state); 2) Functional validation of GTP-binding using radioactive or fluorescent GTP analogs; 3) GTP hydrolysis assays to confirm catalytic activity. Antibody development can proceed as the safest application. Avoid all interaction and inhibitor screening studies until proper folding and GTP-binding capability are confirmed. For reliable GNAQ functional studies, obtain the protein from eukaryotic expression systems capable of proper folding and post-translational modifications. Always include appropriate controls, such as known GTPase standards and validated binding partners.
