Recombinant Rhodopseudomonas palustris UPF0060 membrane protein RPB_2370 (RPB_2370)
Description
Functional Insights and Potential Roles
While RPB_2370 is annotated as a UPF0060 membrane protein, its exact biological function remains uncharacterized. Membrane proteins typically participate in processes such as:
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Signal transduction: Mediating interactions between extracellular and intracellular environments.
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Transport: Facilitating the movement of ions or molecules across membranes.
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Enzymatic activity: Catalyzing biochemical reactions at the membrane interface.
The UPF0060 family is associated with unannotated proteins, suggesting RPB_2370 may belong to a novel functional class. Its classification as a membrane protein aligns with broader studies on R. palustris, which emphasize the bacterium’s metabolic versatility and membrane-bound processes like photosynthesis and carbon fixation .
Production and Purification Protocols
RPB_2370 is produced via recombinant expression in E. coli, leveraging the bacterium’s robust protein synthesis machinery. Post-expression, purification involves:
Product Specs
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Target Background
KEGG: rpb:RPB_2370
STRING: 316058.RPB_2370
Q&A
Structural and Functional Characterization
How does the recombinant RPB_2370 membrane protein interact with hydrophobic compounds like PFOA?
RPB_2370, a UPF0060 membrane protein, exhibits hydrophobic interactions with perfluorooctanoic acid (PFOA) due to its membrane-embedded nature. Studies demonstrate that PFOA incorporates into lipid bilayers, expanding membrane fluidity until saturation, which correlates with RPB_2370’s potential role in membrane stability . Researchers should assess PFOA uptake kinetics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and transmission electron microscopy (TEM) to visualize membrane structural changes under varying PFOA concentrations .
What experimental approaches can resolve discrepancies in PFOA uptake quantification?
Discrepancies often arise from adsorption to cell membranes or vessel surfaces. To resolve this, implement:
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Mass balance analysis: Track PFOA partitioning between media, cells, and surfaces using LC-MS/MS .
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Surface charge measurements: Use a Zetasizer to monitor electrostatic interactions between RPB_2370 and PFOA under high ion conditions .
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Control experiments: Include lysed cells to distinguish active uptake from passive adsorption .
Expression and Purification Challenges
Why does recombinant RPB_2370 often yield low expression in heterologous systems?
Low yields may stem from:
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Incompatible codon usage: Rhodopseudomonas palustris’ codon bias differs from E. coli or yeast systems. Codon optimization (e.g., optimizing G+C content) is critical .
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Membrane protein instability: Use detergents (e.g., DDM or CHAPS) and stabilizing agents (e.g., glycerol) during purification .
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Proper folding: Test refolding protocols using chaperones or in vitro systems if inclusion bodies form .
How to optimize purification of RPB_2370 from inclusion bodies?
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Denaturation and refolding: Use urea gradients (8M → 0M) with redox systems (e.g., GSH/GSSG) .
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Affinity chromatography: Use His-tagged constructs for nickel-nitrilotriacetic acid (Ni-NTA) purification .
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Quality control: Validate via SDS-PAGE and Western blotting with anti-His tags .
Toxicity and Adaptive Mechanisms
What growth patterns indicate R. palustris’ adaptation to PFOA toxicity?
In PFOA-spiked media, R. palustris exhibits diauxic growth:
How to model PFOA’s dose-dependent toxicity for RPB_2370 studies?
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Serial dilution assays: Test PFOA concentrations from 0.78 to 200 ppm, measuring OD660 over 5 days .
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TEM analysis: Visualize membrane integrity post-PFOA exposure .
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Ion chromatography: Monitor anion release to assess membrane damage .
Advanced Research Applications
How to leverage RPB_2370’s dehalogenase activity for PFAS bioremediation?
While RPB_2370 itself lacks direct dehalogenase function, its membrane integration enables:
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Synergistic systems: Co-express with dehalogenases (e.g., fluoroacetate dehalogenase) to enhance PFAS degradation .
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Biosensor development: Engineer RPB_2370 mutants for PFAS detection via fluorescence or electrochemical signals.
What data suggest RPB_2370’s role in membrane fluidity regulation?
PFOA incorporation into lipid bilayers increases fluidity until saturation, as observed via:
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LC-MS/MS: ~44% PFOA removal after 20 days, followed by release .
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Surface charge measurements: Reduced electrostatic repulsion under high ion conditions .
Troubleshooting Experimental Design
Why do PFOA uptake rates vary between live and lysed R. palustris cultures?
Live cultures show transient PFOA removal (44% at 20 days), while lysed cells release PFOA immediately. This discrepancy arises from:
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Active transport: Live cells temporarily sequester PFOA via membrane interactions .
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Passive adsorption: Lysed cells release bound PFOA instantly .
How to validate RPB_2370’s structural integrity post-purification?
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Circular dichroism (CD) spectroscopy: Confirm α-helical/membrane-integrated structure.
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Native mass spectrometry: Assess oligomeric state.
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Functional assays: Test PFOA binding via isothermal titration calorimetry (ITC).
Frontier Research Directions
What unexplored aspects of RPB_2370 warrant further investigation?
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Evolutionary conservation: Compare RPB_2370 homologs across phototrophic bacteria for PFAS resistance mechanisms.
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Membrane remodeling: Study PFOA-induced lipid composition changes using lipidomics.
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Synthetic biology: Engineer RPB_2370 variants with enhanced PFAS binding affinity for bioremediation.
