Recombinant Polaromonas sp. UPF0391 membrane protein Bpro_0066 (Bpro_0066)

What is UPF0391 membrane protein Bpro_0066 and what organism does it originate from?

Bpro_0066 is a membrane protein belonging to the UPF0391 (Uncharacterized Protein Family 0391) found in Polaromonas sp. strain JS666 (ATCC BAA-500). The protein is encoded within the 5.2 Mb circular chromosome of this bacterium, which also contains two circular plasmids (pPol360, 360 kb; and pPol338, 338 kb) . Polaromonas sp. strain JS666 was originally isolated from granular activated carbon sampled at a pump-and-treat plant in Germany . This bacterium belongs to the class Betaproteobacteria and is notable for its potential applications in bioremediation, particularly for degrading various compounds including n-alkanes, cyclic alkanes, haloalkanes, and haloacids .

What are the optimal storage and handling conditions for recombinant Bpro_0066?

For optimal stability and activity maintenance, recombinant Bpro_0066 should be stored according to these specific guidelines:

• Storage buffer: Tris-based buffer containing 50% glycerol, specifically optimized for this protein

• Long-term storage: -20°C, with extended storage recommended at -20°C or -80°C

• Working aliquots: Store at 4°C for up to one week

• Important note: Repeated freezing and thawing is not recommended as it may compromise protein integrity

What expression systems have been successfully used for recombinant Bpro_0066 production?

Based on available data, recombinant Bpro_0066 has been successfully expressed in E. coli expression systems . The recombinant protein has been produced with a His-tag, which facilitates purification through affinity chromatography methods . While specific expression optimization parameters are not detailed in the available literature, standard approaches for membrane protein expression in E. coli would likely include:

• Selection of specialized E. coli strains designed for membrane protein expression

• Temperature optimization (typically lower temperatures around 16-25°C)

• Induction conditions optimization (IPTG concentration and timing)

• Potential co-expression with chaperones to aid proper folding

Given the small size of the protein (61 amino acids), relatively high expression yields should be achievable with properly optimized conditions.

How can researchers verify the identity and integrity of purified recombinant Bpro_0066?

Verification of recombinant Bpro_0066 identity and integrity should employ multiple complementary analytical techniques:

For membrane proteins like Bpro_0066, particular attention should be paid to the detergent environment during analysis, as this can significantly impact the protein's behavior in various analytical techniques.

What purification strategies are most effective for isolating recombinant Bpro_0066?

Purification of membrane proteins like Bpro_0066 requires specialized approaches that maintain protein stability while in a detergent-solubilized state:

• Membrane Isolation: Following cell lysis, membrane fractions containing Bpro_0066 should be isolated through differential centrifugation

• Solubilization: Careful selection of detergents (such as DDM, LDAO, or OG) is crucial for extracting the protein from membranes while maintaining native conformation

• Affinity Chromatography: His-tagged Bpro_0066 can be purified using immobilized metal affinity chromatography (IMAC) with Ni-NTA or similar resins

• Size Exclusion Chromatography: A final polishing step to remove aggregates and achieve high purity

For a small membrane protein like Bpro_0066, achieving high purity is essential for subsequent structural and functional studies.

What is currently known about the function of UPF0391 family membrane proteins like Bpro_0066?

Polaromonas sp. strain JS666 has been studied for its remarkable ability to degrade various compounds and resist toxic elements like mercury and arsenic . The genome contains genes involved in the degradation of n-alkanes, cyclic alkanes, cyclic alcohols, haloalkanes, and haloacids . The presence of Bpro_0066 within this organism suggests several possible functional hypotheses:

• It may play a role in membrane adaptation to environmental stressors

• It could be involved in transport or sensing mechanisms related to xenobiotic compounds

• It might contribute to the unique metabolic capabilities of this bacterium

Methodological approaches to elucidate function could include gene knockout studies, comparative expression analysis under various stress conditions, and protein-protein interaction studies.

How might Bpro_0066 contribute to cold adaptation in Polaromonas species?

While specific information about Bpro_0066's role in cold adaptation is not explicitly stated in the available literature, this represents an intriguing research question given that:

• Many Polaromonas species are psychrotolerant bacteria found in cold environments, including Arctic and Antarctic glaciers

• Membrane proteins often play crucial roles in adaptation to low temperatures by modulating membrane fluidity and permeability

Experimental approaches to investigate this potential function could include:

What structural biology approaches would be most suitable for studying Bpro_0066?

As a small membrane protein (61 amino acids), Bpro_0066 presents both challenges and opportunities for structural characterization:

• NMR Spectroscopy: Perhaps the most promising approach for a protein of this size. Would require:

  • Expression in isotope-enriched media (13C, 15N)

  • Optimization of detergent or lipid environments

  • Selection of appropriate pulse sequences for membrane protein analysis

• X-ray Crystallography: Challenging for membrane proteins but possible with:

  • Lipidic cubic phase (LCP) crystallization

  • Addition of crystallization chaperones or antibody fragments

  • High-throughput screening of crystallization conditions

• Cryo-Electron Microscopy: Traditionally challenging for proteins <50 kDa, but recent advances with:

  • Scaffold proteins to increase effective size

  • New detection technologies improving resolution

• Computational Modeling: Particularly valuable given the small size:

  • Homology modeling if structural homologs exist

  • Ab initio modeling becoming increasingly accurate for smaller proteins

  • Molecular dynamics simulations in membrane environments

What protocols exist for studying membrane protein interactions involving Bpro_0066?

Investigating potential interaction partners of Bpro_0066 requires specialized approaches suited to membrane proteins:

• Affinity Purification-Mass Spectrometry (AP-MS):

  • Using His-tagged Bpro_0066 as bait in Polaromonas lysates

  • Maintaining appropriate detergent concentrations throughout

  • Identifying co-purifying proteins through LC-MS/MS

• In vivo Crosslinking:

  • Chemical crosslinkers with membrane permeability

  • Photoactivatable crosslinkers for higher specificity

  • Crosslinking in native Polaromonas followed by affinity purification

• Bacterial Two-Hybrid Systems:

  • Modified systems designed for membrane protein analysis

  • BACTH (Bacterial Adenylate Cyclase Two-Hybrid) system

• Reconstitution Studies:

  • Co-reconstitution of purified Bpro_0066 with candidate partners

  • Functional assays to detect physical and functional interactions

What approaches are recommended for generating antibodies against small membrane proteins like Bpro_0066?

Generating specific antibodies against small membrane proteins requires careful consideration:

• Epitope Selection:

  • For a 61-amino acid protein like Bpro_0066, identify hydrophilic regions

  • Use epitope prediction algorithms that account for membrane topology

  • Consider both N-terminal and C-terminal regions which may be more accessible

• Immunization Strategies:

  • Synthetic peptide approach: Design peptides (12-20 aa) from predicted antigenic regions

  • Recombinant protein approach: Immunize with purified Bpro_0066 in detergent micelles

  • Consider carrier proteins to enhance immunogenicity of small peptides

• Validation Methods:

  • ELISA against immunizing antigen

  • Western blotting against recombinant Bpro_0066

  • Immunofluorescence in Polaromonas sp. expressing Bpro_0066

  • Control tests with Bpro_0066 knockout strains

How can researchers investigate the role of Bpro_0066 in plasmid biology of Polaromonas sp. strain JS666?

The Polaromonas sp. strain JS666 genome includes a chromosome and two large plasmids (pPol360 and pPol338) . While Bpro_0066 is chromosomally encoded, investigating its potential role in plasmid biology represents an interesting research direction:

• Comparative Genomics:

  • Analyze the genomic context of Bpro_0066 and potential paralogs

  • Compare with related genes in other Polaromonas strains with different plasmid compositions

• Expression Analysis:

  • Monitor Bpro_0066 expression levels in plasmid-cured versus wild-type strains

  • Investigate co-expression patterns with plasmid-encoded genes

• Protein-DNA Interaction Studies:

  • Chromatin immunoprecipitation (ChIP) to identify potential DNA binding

  • Electrophoretic mobility shift assays (EMSA) with purified protein

• Conjugation Experiments:

  • Create Bpro_0066 knockout strains and assess impact on conjugative transfer

  • Measure plasmid stability in the presence/absence of functional Bpro_0066

What strategies can overcome low expression yields of recombinant Bpro_0066?

Low expression is a common challenge when working with membrane proteins. For Bpro_0066, researchers might consider:

• Codon Optimization: Adapt the gene sequence for optimal expression in the host organism

• Expression Tags: Test different fusion partners (MBP, SUMO, Trx) to improve solubility

• Specialized Strains: C41(DE3), C43(DE3), or Lemo21(DE3) E. coli strains specifically designed for membrane protein expression

• Culture Conditions: Lower temperature (16-20°C), reduced inducer concentration, and specialized media formulations

How can researchers address aggregation issues during purification of Bpro_0066?

Aggregation is a significant challenge when working with membrane proteins. For Bpro_0066, consider these methodological approaches:

• Detergent Screening: Systematically test different detergent types and concentrations

• Buffer Optimization: Adjust pH, ionic strength, and glycerol content to enhance stability

• Additives: Include specific lipids or cholesterol that might stabilize the native conformation

• Temperature Control: Maintain samples at 4°C throughout purification to minimize aggregation

• Concentration Techniques: Use gentle methods like dialysis against PEG rather than centrifugal concentration

What genomic approaches could reveal the evolutionary significance of Bpro_0066?

Understanding the evolutionary context of Bpro_0066 could provide insights into its function:

• Phylogenetic Analysis:

  • Construct phylogenetic trees of UPF0391 family proteins

  • Map presence/absence patterns across bacterial lineages

  • Correlate with ecological niches and metabolic capabilities

• Synteny Analysis:

  • Examine conservation of genomic context around Bpro_0066

  • Identify co-evolved gene clusters that might suggest functional relationships

• Selection Pressure Analysis:

  • Calculate dN/dS ratios to identify regions under purifying or positive selection

  • Identify conserved residues that might be functionally critical

• Horizontal Gene Transfer Assessment:

  • Determine if Bpro_0066 shows evidence of horizontal acquisition

  • Evaluate if its presence correlates with specific plasmid types across Polaromonas species

How might advanced computational approaches contribute to understanding Bpro_0066 function?

Computational methods offer powerful approaches to predict function:

• AlphaFold2/RoseTTAFold Structure Prediction:

  • Generate high-confidence structural models

  • Compare with known structures to identify potential functional homologs

• Molecular Dynamics Simulations:

  • Model behavior in membrane environments

  • Identify potential binding pockets or interaction interfaces

• Co-evolution Analysis:

  • Identify potentially interacting proteins through correlated mutations

  • Predict functional partners based on evolutionary coupling

• Systems Biology Integration:

  • Incorporate Bpro_0066 into metabolic models of Polaromonas sp.

  • Predict phenotypic consequences of gene deletion through flux balance analysis