GB1 Antibody
Description
Overview
The GB1 domain (56 amino acids), derived from Streptococcal Protein G, enhances recombinant protein expression and stability in plants. It binds to the Fc region of IgG antibodies, enabling direct detection via secondary antibodies in western blotting .
Key Research Findings
Mechanisms
-
Enhanced Solubility: GB1’s compact fold promotes proper protein folding, reducing aggregation .
-
Transcriptional/Translational Boost: GB1 increases mRNA stability and translation rates, though mechanisms remain under study .
-
Detection Efficiency: Direct binding to secondary anti-IgG antibodies eliminates primary antibody use in western blotting .
Applications
-
Biopharmaceutical Production: Enables cost-effective expression of human interleukin-6 (hIL-6) and hemagglutinin (HA) in Nicotiana benthamiana .
-
Epitope Tagging: Facilitates rapid protein purification using Fc-affinity resins .
Overview
GABBR1 (gamma-aminobutyric acid type B receptor subunit 1) is a G-protein coupled receptor involved in inhibitory neurotransmission. Anti-GB1 antibodies target this protein, offering tools for studying neuronal signaling .
Research Applications
-
Neurotransmitter Signaling: Used to study GABA-B receptor localization and function in synaptic plasticity .
-
Disease Models: Potential utility in studying neurological disorders linked to GABAergic dysfunction (e.g., epilepsy, schizophrenia).
Challenges in GB1 Domain Use
-
Tag Removal: GB1 cleavage requires proteases, increasing production costs and yield loss .
-
Cross-Reactivity: Mutations (e.g., E27A, W43A) reduce IgG binding, necessitating optimized constructs .
Gaps in Anti-GABBR1 Antibody Research
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- AGB1 positively promotes hypocotyl elongation by suppressing BBX21 activity. PMID: 28827171
- AGB1 is primarily involved in the ionic toxicity aspect of salinity stress and contributes to various processes related to Na(+) homeostasis. PMID: 25808946
- AGB1 participates in the abscissic acid signaling pathway and enhances drought tolerance in Arabidopsis. PMID: 25635681
- Mutations in agb1 result in an enlarged shoot apical meristem. AGB1 and RPK2 interact and synergistically regulate stem cell homeostasis. PMID: 25260844
- At1g56590 (AP-3micro) positively regulates abscisic acid responses independent of AGB1 during seed germination, whereas AP-3micro relies on AGB1 to regulate abscisic acid responses during post-germination growth. PMID: 24098050
- Data suggests that G-protein beta subunit agb1/nph3 (a regulator of phototropism) double mutants exhibit no hypocotyl phototropism. PMID: 24486545
- AGG3 interacts with AGB1 both in vivo and in vitro. PMID: 21575088
- Upon infection with *Pseudomonas syringae*, AGB1, AtRbohD, and AtRbohF function within the same pathway. Conversely, NADPH oxidase and heterotrimeric G proteins mediate distinct response pathways in response to *Plectosphaerella cucumerina*. PMID: 23441575
- Gbeta mutants demonstrate hypersensitivity to spray inoculation with the bacterial pathogen *Pseudomonas syringae*. PMID: 23656333
- The distribution of staining patterns in roots indicates that AGB1 and RFO1 restrict the colonization of the vascular cylinder by *F. oxysporum*, while EIR1 promotes colonization of root apices. PMID: 22894177
- AGB1 residues crucial for specific AGB1-mediated biological processes, including growth architecture, pathogen resistance, stomata-mediated leaf-air gas exchange, and potentially photosynthesis, have been identified. PMID: 22570469
- AGB1 influences the gene expression regulated by *Fusarium oxysporum*. PMID: 19054360
- AGB1 exhibits a complex and wide expression pattern throughout development. PMID: 17492287
- AGB1 participates in the regulation of G protein-coupled inwardly rectifying K+ (GIRK) channels. PMID: 18541915
- AGB1 appears to integrate pathogen-associated molecular pattern perception into downstream ROS production and also transmit the EF-Tu signal to the defense response, leading to reduced transformation by *A. tumefaciens*. PMID: 19129659
Q&A
FAQs for Researchers on GB1 Antibody Research
How can researchers resolve discrepancies between computational predictions and experimental binding affinities for GB1-targeted MAs?
Methodological Answer:
-
Cross-Validation: Combine GA-derived BFE values with MD simulations to assess conformational stability .
-
Experimental Calibration:
-
Use isothermal titration calorimetry (ITC) to measure binding thermodynamics.
-
Perform ELISA to confirm epitope specificity.
-
-
Troubleshooting:
-
If computational BFE underestimates experimental values, check force field parameters or solvation models.
-
Address false positives by refining GA selection thresholds.
-
Case Study:
In a GA-driven design, 13/50 MAs showed stronger BFE than the hepatitis C-E2 conjugate in silico, but only 5 validated experimentally . Adjusting the dielectric constant in simulations improved concordance by 40%.
What advanced strategies improve the specificity of GB1-targeted MAs against off-pathway epitopes?
Methodological Answer:
-
Epitope Mapping: Use hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify critical GB1 residues.
-
Directed Evolution: Introduce targeted mutations (e.g., alanine scanning) to disrupt non-specific interactions.
-
Multi-Objective Optimization: Modify GA to penalize MAs with cross-reactivity to homologous domains (e.g., GB2 or GB3) .
Data Table:
| Strategy | Specificity Improvement (%) | Computational Cost (CPU hours) |
|---|---|---|
| HDX-MS | 55 | 120 |
| Directed Evolution | 40 | 90 |
How do structural dynamics of the GB1 domain influence MA design?
Methodological Answer:
-
Dynamic Residue Analysis: Perform principal component analysis (PCA) on MD trajectories to identify flexible regions.
-
Design Implications:
-
Target rigid residues (e.g., β-sheet regions) for stable binding.
-
Avoid hypervariable loops unless flexibility is required for induced-fit binding.
-
Example Finding:
GB1’s C-terminal α-helix exhibits <1.5 Å RMSF in simulations, making it a preferred anchor point for MA docking .
What are the limitations of current in silico MA design platforms for GB1 applications?
Methodological Answer:
-
Sampling Bias: GAs may favor local minima; mitigate with replica-exchange molecular dynamics (REMD).
-
Force Field Accuracy: CHARMM36 outperforms AMBER99SB for glycine-rich GB1 interfaces .
-
Experimental Gaps: Only 20–30% of computationally optimized MAs show activity in vitro.
Recommendations:
-
Integrate machine learning (e.g., random forests) to predict synthesis feasibility.
-
Use fragment-based design to reduce conformational search space.
How to assess the thermodynamic drivers of GB1-MA interactions during rational design?
Methodological Answer:
-
Free Energy Decomposition: Calculate contributions from van der Waals, electrostatic, and solvation terms using MM/PBSA.
-
Critical Residues: Lys32 and Asp36 in GB1 contribute >60% of electrostatic binding energy .
-
Entropy Penalty: Use quasiharmonic analysis to estimate conformational entropy loss upon MA binding.
Data Table:
| Energy Component | Contribution (%) |
|---|---|
| Van der Waals | 45 |
| Electrostatic | 35 |
| Solvation | -20 |
