Recombinant Burkholderia xenovorans UPF0060 membrane protein Bxeno_B1021 (Bxeno_B1021)

What is Bxeno_B1021 and what are its basic structural features?

Bxeno_B1021 (also known as Bxe_B1996) is a UPF0060 family membrane protein from Paraburkholderia xenovorans (formerly classified as Burkholderia xenovorans). The full-length protein consists of 106 amino acids with the sequence: MKTFLLYAVTAVAEIVGCYLPWRWLKEGGSIWLLVPGALSLALFAWLLTLHGTAAGRVYAAYGGVYVAVAIAWLWCVDKVRPTLWDAAGVVFTLAGMAIIAFQPRV . As suggested by its hydrophobic amino acid composition, Bxeno_B1021 is an integral membrane protein with multiple transmembrane regions. The protein is identified in the UniProt database with the accession number Q13PK0 .

When expressed recombinantly, the protein is typically fused with an N-terminal His-tag to facilitate purification using immobilized metal affinity chromatography (IMAC). The presence of multiple hydrophobic domains suggests that this protein is embedded within cellular membranes, which influences both experimental approaches and functional hypotheses.

What expression systems are commonly used for recombinant Bxeno_B1021 production?

For research applications, Bxeno_B1021 is primarily expressed in E. coli expression systems optimized for membrane protein production . The selection of an appropriate expression system is critical due to the membrane-embedded nature of the protein, which can lead to toxicity, aggregation, or misfolding in standard expression systems.

When working with this protein, researchers should consider the following methodological approaches:

• Using specialized E. coli strains (such as C41/C43 or BL21-AI) designed for membrane protein expression

• Optimizing induction conditions (lower temperatures, reduced IPTG concentrations)

• Supplementing with specific membrane-stabilizing additives

• Employing fusion partners that enhance membrane insertion and stability

While E. coli remains the predominant expression system as documented in the literature, advanced research may explore alternative expression systems such as insect cells or yeast when native-like membrane composition is required for structural or functional studies .

What are the optimal storage and reconstitution conditions for recombinant Bxeno_B1021?

Maintaining structural integrity and functionality of Bxeno_B1021 requires careful attention to storage and reconstitution protocols. Based on experimental evidence, the following approach is recommended:

Storage Protocol:

• Store lyophilized protein at -20°C/-80°C upon receipt

• For working solutions, store at 4°C for no more than one week

• Avoid repeated freeze-thaw cycles as they significantly diminish protein integrity

Reconstitution Protocol:

• Briefly centrifuge the vial prior to opening to collect contents

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (typically 50%) for long-term storage

• Aliquot to minimize freeze-thaw cycles and store at -20°C/-80°C

The reconstitution buffer composition may need modification depending on downstream applications. Researchers investigating protein-lipid interactions or structural studies may require specific detergents or lipid mixtures to maintain native conformation during reconstitution.

How can researchers optimize membrane extraction and purification protocols for Bxeno_B1021?

Efficient extraction and purification of membrane proteins like Bxeno_B1021 requires specialized approaches to maintain structural integrity while achieving high purity. Recommended methodology includes:

Extraction Protocol:

• Harvest cells expressing Bxeno_B1021 at optimal density

• Resuspend in buffer containing protease inhibitors

• Disrupt cell membranes using methods such as sonication or high-pressure homogenization

• Isolate membrane fractions through differential centrifugation

• Solubilize membrane proteins using appropriate detergents (typically non-ionic detergents like DDM, LDAO, or OG)

Purification Considerations:

• Leverage the His-tag for IMAC purification

• Consider detergent exchange during purification to optimize stability

• Implement size exclusion chromatography as a final polishing step

• Monitor protein quality using analytical techniques (e.g., SDS-PAGE, Western blot, mass spectrometry)

Researchers working with this protein should be aware that extraction efficiency is highly dependent on detergent selection. Preliminary screening of multiple detergents is often necessary to identify optimal conditions for specific downstream applications .

What techniques are recommended for studying Bxeno_B1021 localization and membrane topology?

Understanding the precise membrane topology and subcellular localization of Bxeno_B1021 is essential for functional characterization. Advanced techniques recommended for these studies include:

Membrane Topology Analysis:

• Cysteine scanning mutagenesis: Systematic replacement of residues with cysteine followed by accessibility testing with membrane-impermeable thiol-reactive reagents

• Protease protection assays: Controlled proteolytic digestion with domain-specific antibody detection

• Fluorescence approaches: Position-specific fluorescent labeling combined with quencher accessibility studies

Localization Studies:

• Immunofluorescence microscopy: Using antibodies against the His-tag or the protein itself

• Fluorescent protein fusions: Creating GFP/RFP fusions for live-cell imaging

• Subcellular fractionation: Paired with Western blot analysis for biochemical verification

For in vivo studies, researchers might consider adapting the glycocapture method described for blood-brain barrier proteins, which demonstrates high specificity for cell surface proteins, especially those that are glycosylated . This approach could be particularly valuable if Bxeno_B1021 is found to have glycosylation modifications in its native context.

How can researchers investigate potential binding partners and protein-protein interactions of Bxeno_B1021?

Identifying binding partners and protein-protein interactions is crucial for elucidating the functional role of Bxeno_B1021. Several complementary approaches are recommended:

In vitro Interaction Studies:

• Pull-down assays: Utilizing the His-tag on recombinant Bxeno_B1021

• Surface Plasmon Resonance (SPR): For quantitative binding kinetics

• Isothermal Titration Calorimetry (ITC): For thermodynamic parameters of interactions

In vivo Interaction Studies:

• Proximity labeling approaches: BioID or APEX2 fusions to identify proximal proteins

• Crosslinking Mass Spectrometry (XL-MS): For capturing transient interactions

• Förster Resonance Energy Transfer (FRET): For monitoring interactions in living cells

For membrane proteins like Bxeno_B1021, maintaining the native membrane environment is particularly challenging. Researchers should consider reconstituting the protein in nanodiscs or liposomes for interaction studies to preserve physiologically relevant conformations and interaction interfaces .

What approaches can identify the functional role of Bxeno_B1021 in bacterial physiology?

Despite being classified as a UPF0060 family protein with unknown function, several systematic approaches can help elucidate the physiological role of Bxeno_B1021:

Genetic Approaches:

• Gene knockout/knockdown: Analyzing phenotypic consequences of Bxeno_B1021 deletion

• Complementation studies: Rescuing knockout phenotypes with wild-type and mutant variants

• Conditional expression systems: Investigating effects of protein depletion or overexpression

Physiological Characterization:

• Stress response assays: Testing sensitivity to various environmental stressors

• Membrane integrity analyses: Assessing permeability and potential transporter functions

• Metabolomic profiling: Identifying metabolic pathways affected by Bxeno_B1021 manipulation

Since search result indicates that Bxeno_B1021 participates in several biological pathways, researchers should consider systematic phenotypic screening approaches. Cross-referencing experimental findings with bioinformatic predictions can narrow down potential functional hypotheses for targeted investigation.

How can researchers address the challenges of structural studies for Bxeno_B1021?

Determining the three-dimensional structure of membrane proteins like Bxeno_B1021 presents significant challenges. The following approaches represent current best practices:

Sample Preparation Strategies:

• Detergent screening: Systematic evaluation of detergents for stability and homogeneity

• Lipid cubic phase crystallization: For maintaining membrane-like environment

• Nanodiscs or amphipols: For detergent-free stabilization

Structural Determination Methods:

• X-ray crystallography: Requires high-quality crystals, challenging for membrane proteins

• Cryo-electron microscopy: Increasingly powerful for membrane protein structures

• Nuclear Magnetic Resonance (NMR): Suitable for smaller membrane proteins or domains

Complementary Approaches:

• Molecular dynamics simulations: For refining structures and studying dynamics

• Hydrogen-deuterium exchange mass spectrometry: For probing conformational changes

• Cross-linking studies: For constraining structural models

Given the relatively small size of Bxeno_B1021 (106 amino acids), solution NMR might be feasible if sufficient quantities of isotopically-labeled protein can be produced in a suitable membrane mimetic environment .

How does Bxeno_B1021 compare to homologous proteins in other bacterial species?

Understanding the evolutionary context of Bxeno_B1021 can provide valuable insights into its function. A comprehensive comparative analysis should include:

Sequence Analysis:

Structural Conservation:

• Analyze predicted transmembrane topology across homologs

• Identify conserved motifs or residues for targeted mutagenesis

• Examine co-evolution patterns to predict functional interfaces

Genomic Context Analysis:

• Investigate neighboring genes across species

• Identify conserved operon structures

• Look for co-occurrence patterns with functionally characterized genes

This comparative approach can reveal conserved features that have been maintained through evolutionary pressure, suggesting functional importance. Combined with experimental data, these analyses can guide hypothesis generation for functional studies .

What bioinformatic tools and databases are most useful for analyzing Bxeno_B1021?

To conduct comprehensive bioinformatic analysis of Bxeno_B1021, researchers should utilize a combination of specialized tools and databases:

Sequence Analysis Tools:

• BLAST/PSI-BLAST: For identifying homologs across species

• Multiple Sequence Alignment tools: MUSCLE, CLUSTAL, T-Coffee

• Phylogenetic analysis: RAxML, MrBayes, PhyML

Structural Prediction Resources:

• Transmembrane topology: TMHMM, TOPCONS, Phobius

• Secondary structure: PSIPRED, JPred

• Tertiary structure: AlphaFold2, RoseTTAFold, I-TASSER

Functional Prediction Tools:

• InterProScan: For domain and family identification

• STRING: For protein-protein interaction networks

• KEGG/BioCyc: For metabolic pathway mapping

Specialized Membrane Protein Resources:

• MemProtMD: For membrane protein simulations

• OPM database: For orientation of proteins in membranes

• PDBTM: Transmembrane protein structure database

By integrating results from these resources, researchers can develop testable hypotheses about structure-function relationships, potential interaction partners, and physiological roles of Bxeno_B1021 .

How can researchers overcome common challenges in expression and purification of Bxeno_B1021?

Membrane proteins like Bxeno_B1021 present specific challenges in recombinant expression and purification. The following troubleshooting approaches address common issues:

Expression Challenges:

Purification Challenges:

Researchers should implement systematic optimization strategies rather than changing multiple variables simultaneously, documenting conditions that improve protein quality and yield .

What quality control methods are essential for validating recombinant Bxeno_B1021?

Ensuring the quality of purified Bxeno_B1021 is critical for reliable downstream experiments. A comprehensive quality control workflow should include:

Basic Characterization:

• SDS-PAGE: For purity assessment (>90% recommended)

• Western blotting: For identity confirmation

• Mass spectrometry: For accurate mass determination and sequence verification

Functional Validation:

• Circular dichroism: For secondary structure assessment

• Fluorescence spectroscopy: For tertiary structure evaluation

• Size exclusion chromatography: For oligomeric state analysis

Stability Assessment:

• Thermal shift assays: For determining thermal stability

• Time-course activity/structure measurements: For monitoring stability over time

• Detergent/lipid optimization: For identifying stabilizing conditions

For membrane proteins like Bxeno_B1021, additional specialized quality control may include reconstitution into proteoliposomes or nanodiscs to verify proper membrane insertion and functional state. Researchers should establish acceptance criteria for each quality parameter before proceeding to functional or structural studies .

What emerging technologies could advance our understanding of Bxeno_B1021?

Several cutting-edge methodologies show promise for deeper characterization of membrane proteins like Bxeno_B1021:

Advanced Structural Approaches:

• Microcrystal electron diffraction (MicroED): For structure determination from nano-sized crystals

• Integrative structural biology: Combining multiple data types (cross-linking, HDX-MS, cryo-EM) for comprehensive models

• Serial crystallography: Using X-ray free-electron lasers for room-temperature structures

Functional Genomics:

• CRISPRi/CRISPRa systems: For precise control of gene expression in native contexts

• RNA-seq and Ribo-seq: For transcriptomic and translational impacts of Bxeno_B1021 modulation

• Metabolic flux analysis: For determining effects on cellular metabolism

Single-molecule approaches:

• Single-molecule FRET: For conformational dynamics

• High-speed AFM: For visualizing structural changes in real-time

• Nanopore recording: If Bxeno_B1021 has channel or transport functions

The novel glycocapture approach described in search result could potentially be adapted for studying Bxeno_B1021 in its native membrane environment, providing insights into its accessibility and potential interactions with extracellular factors .

How might research on Bxeno_B1021 contribute to broader understanding of bacterial membrane biology?

Investigation of Bxeno_B1021 has potential implications for several important areas of bacterial membrane biology:

Fundamental Membrane Protein Biology:

• Insights into membrane protein folding and stability

• Understanding of protein-lipid interactions in bacterial membranes

• Models for membrane protein evolution and specialization

Bacterial Physiology:

• Potential roles in membrane organization or compartmentalization

• Contributions to stress response or environmental adaptation

• Involvement in signaling across bacterial membranes

Comparative Microbiology:

• Functional conservation across bacterial species

• Species-specific adaptations of membrane proteomes

• Evolution of specialized membrane functions

While the specific function of Bxeno_B1021 remains to be fully characterized, systematic investigation using the approaches outlined in this FAQ will contribute to our understanding of this protein family and potentially reveal new aspects of bacterial membrane biology .