Recombinant Coxiella burnetii UPF0133 protein CBU_0656 (CBU_0656)

What is UPF0133 protein CBU_0656 and why is it significant for research?

CBU_0656 is a hypothetical protein encoded by the Coxiella burnetii genome at position 601425-601757 on the negative strand. It consists of 110 amino acids and has been designated as part of the UPF0133 protein family (Uncharacterized Protein Family 0133) .

The significance of CBU_0656 lies in its potential role in C. burnetii pathogenesis, the bacterium responsible for Q fever in humans. As a hypothetical protein, its function remains largely uncharacterized, making it an important target for research into novel bacterial proteins that may contribute to virulence mechanisms or potential diagnostic applications .

What is currently known about the genomic context of CBU_0656?

CBU_0656 is located within the C. burnetii RSA 493 genome with the following characteristics:

• Genomic position: 601425..601757

• Strand: Negative

• Length: 110 amino acids

• Protein ID: 29653994

• G+C content: 42.04%

The protein is situated in proximity to other genes including:

• CBU_0655 (hypothetical protein, positions 600472..601422)

• CBU_0657 (recombination protein RecR, positions 601771..602376)

This genomic context suggests potential functional relationships with neighboring proteins, particularly RecR, which is involved in DNA recombination and repair processes .

What expression systems are recommended for producing recombinant CBU_0656?

For optimal expression of recombinant CBU_0656, the following systems are recommended:

For initial characterization studies, E. coli or yeast systems are recommended for their efficiency. For functional studies where protein activity and proper folding are critical, insect or mammalian expression systems may be preferable despite their higher cost and complexity .

How should I design an experimental protocol to express and purify recombinant CBU_0656?

A robust experimental protocol for CBU_0656 expression and purification should include:

Expression Protocol:

• Clone the CBU_0656 gene into an appropriate expression vector with a purification tag (His-tag recommended based on similar protein studies)

• Transform into E. coli BL21(DE3) or similar expression strain

• Grow cultures to mid-log phase (OD600 0.6-0.8) in LB or minimal media

• Induce protein expression with IPTG (0.1-1.0 mM) or auto-induction media

• Incubate at reduced temperature (16-25°C) for 16-20 hours to enhance soluble protein yield

Purification Protocol:

• Harvest cells by centrifugation (5,000 × g, 15 min, 4°C)

• Resuspend in lysis buffer (50 mM HEPES pH 8.0, 100 mM NaCl, 1 mM TCEP)

• Lyse cells using sonication or pressure homogenization

• Clarify lysate by centrifugation (20,000 × g, 30 min, 4°C)

• Purify using Ni-NTA affinity chromatography

• Further purify using size exclusion chromatography

• Assess purity using SDS-PAGE

• Store at -80°C in buffer containing 50 mM HEPES pH 8.0, 100 mM NaCl, 1 mM TCEP

This protocol is based on successful approaches used for similar hypothetical proteins and carrier-free recombinant proteins .

What are the critical variables to control in CBU_0656 expression experiments?

When designing experiments for CBU_0656 expression, control the following variables to ensure reproducibility and optimal protein yield:

• Independent Variables:

  • Expression host strain (E. coli BL21(DE3), Rosetta, etc.)

  • Induction method (IPTG concentration, auto-induction)

  • Growth temperature pre- and post-induction

  • Media composition (rich vs. minimal)

  • Vector design (promoter strength, fusion tags)

• Dependent Variables:

  • Protein yield (mg/L culture)

  • Protein solubility (% in soluble fraction)

  • Protein purity (after purification)

  • Protein activity (if functional assays available)

• Extraneous Variables to Control:

  • Bacterial growth phase at induction (standardize OD600)

  • Aeration conditions (consistent shaking speed, flask-to-media volume ratio)

  • Incubation time post-induction

  • Cell lysis methods and buffer composition

A well-designed experiment should include appropriate controls such as uninduced cultures and empty vector transformants to assess background expression and host protein contamination .

How can I assess the quality and stability of purified recombinant CBU_0656?

To assess the quality and stability of purified recombinant CBU_0656:

• Purity Assessment:

  • SDS-PAGE with Coomassie staining (target >95% purity)

  • Western blot using anti-His antibodies (if His-tagged)

  • Mass spectrometry to confirm protein identity and detect modifications

• Stability Assessment:

  • Thermal shift assay (differential scanning fluorimetry) to determine melting temperature

  • Dynamic light scattering to assess homogeneity and aggregation state

  • Size exclusion chromatography to evaluate oligomeric state

  • Stability at different temperatures (4°C, -20°C, -80°C) over time

• Quantification Methods:

  • UV spectroscopy (A280 measurement)

  • Bradford or BCA protein assays

  • Amino acid analysis for absolute quantification

• Storage Recommendations:

  • Store in buffer containing stabilizing agents (reducing agents like TCEP)

  • Avoid repeated freeze-thaw cycles

  • Consider flash-freezing aliquots in liquid nitrogen before -80°C storage

Quality assessment should be performed immediately after purification and at regular intervals during storage to monitor potential degradation.

How can I investigate the potential function of this hypothetical protein?

To investigate the function of CBU_0656, employ a multi-faceted approach:

• Bioinformatics Analysis:

  • Sequence homology searches against characterized proteins

  • Structure prediction using tools like AlphaFold2

  • Conserved domain identification

  • Genomic context analysis (neighboring genes)

• Structural Studies:

  • X-ray crystallography

  • Nuclear magnetic resonance (NMR) spectroscopy

  • Cryo-electron microscopy

  • Circular dichroism to analyze secondary structure

• Protein-Protein Interaction Studies:

  • Yeast two-hybrid screening

  • Pull-down assays with host cell lysates

  • Affinity purification coupled with mass spectrometry (AP-MS)

  • Surface plasmon resonance to measure binding kinetics

• Functional Assays:

  • Gene knockout/knockdown in C. burnetii followed by phenotypic analysis

  • Heterologous expression in host cells to observe localization and effects

  • Transcriptomics to identify co-regulated genes

  • Metabolomics to detect changes in metabolic pathways

These approaches should be performed in parallel to provide complementary lines of evidence for functional characterization.

Could CBU_0656 play a role in C. burnetii pathogenesis or Q fever?

While direct evidence linking CBU_0656 to C. burnetii pathogenesis remains limited, several methodological approaches can investigate this question:

• Comparative Analysis:

  • Compare expression levels of CBU_0656 between virulent and avirulent C. burnetii strains

  • Analyze expression during different stages of infection (early vs. late)

  • Examine conservation across different clinical isolates

• Host Response Studies:

  • Assess immune reactivity by screening patient sera against recombinant CBU_0656

  • Determine if antibodies against CBU_0656 are present in acute or chronic Q fever patients

  • Compare with known immunoreactive C. burnetii proteins (Com1, GroEL)

• Cellular Localization:

  • Determine subcellular localization within C. burnetii using immunogold electron microscopy

  • Assess whether CBU_0656 is secreted or surface-exposed

  • Investigate if CBU_0656 interacts with host cell compartments

• Infection Models:

  • Evaluate virulence of CBU_0656 knockout mutants in cell culture and animal models

  • Assess bacterial replication, vacuole formation, and host cell responses

  • Compare infection kinetics to wild-type strains

Based on studies of other hypothetical C. burnetii proteins, potential roles may include involvement in intracellular survival, modulation of host immune responses, or contribution to the parasitophorous vacuole formation.

How does CBU_0656 compare to other UPF family proteins structurally and functionally?

To compare CBU_0656 with other UPF family proteins:

• Structural Comparison Methods:

  • Generate structural models using AlphaFold2 or similar tools

  • Compare predicted structures with known UPF family structures

  • Analyze conserved structural motifs and potential active sites

  • Use multiple loop permutation (MLP) techniques to identify structural similarities with proteins outside the UPF family

• Sequence Analysis Approaches:

  • Multiple sequence alignment of UPF0133 family members

  • Phylogenetic analysis to determine evolutionary relationships

  • Identification of conserved residues that may be functionally important

  • Analysis of sequence variations specific to pathogenic species

• Functional Comparison Strategies:

  • Complementation studies using other UPF0133 family members

  • Comparative protein-protein interaction profiling

  • Assessment of similar biochemical activities or cellular localization

  • Cross-species functional conservation analysis

What protein interaction networks might CBU_0656 participate in?

To elucidate protein interaction networks involving CBU_0656:

• Experimental Approaches:

  • Affinity purification coupled with mass spectrometry (AP-MS)

  • Proximity-dependent biotin identification (BioID)

  • FRET/BRET-based interaction mapping

  • High-throughput yeast two-hybrid screening against both C. burnetii and host proteins

• Potential Interaction Partners:

  • Based on studies of other C. burnetii proteins, CBU_0656 might interact with:

    • Components of the ubiquitin-proteasome system

    • Autophagy-related proteins

    • Vesicular trafficking components

    • Other bacterial effector proteins

• Network Analysis:

  • Integration of experimental data with existing C. burnetii protein interaction networks

  • Identification of hub proteins and functional clusters

  • Cross-reference with host-pathogen interaction databases

  • Validation of key interactions using co-immunoprecipitation and mutational analysis

Recent studies have revealed that many C. burnetii proteins interact with host proteasome and autophagy machinery, suggesting potential involvement of CBU_0656 in these pathways, particularly given its hypothetical nature and unknown function .

What approaches can be used to determine the three-dimensional structure of CBU_0656?

For determining the 3D structure of CBU_0656:

• X-ray Crystallography Approach:

  • Express CBU_0656 with various tags (His, GST, MBP) to improve solubility

  • Screen multiple crystallization conditions (temperature, pH, precipitants)

  • Consider surface entropy reduction mutations to enhance crystallization

  • Optimize diffraction-quality crystals through microseeding techniques

  • Process diffraction data using appropriate software packages

• NMR Spectroscopy Method:

  • Express isotopically labeled protein (15N, 13C)

  • Perform heteronuclear single quantum coherence (HSQC) experiments

  • Assign backbone and side-chain resonances

  • Generate structural constraints through NOE experiments

  • Calculate and refine 3D structure models

• Cryo-EM Approach:

  • Consider fusion to larger proteins if CBU_0656 is too small for cryo-EM alone

  • Optimize sample preparation and vitrification conditions

  • Collect high-resolution image data

  • Perform single-particle reconstruction

  • Validate resulting models using complementary techniques

• Computational Structure Prediction:

  • Utilize AlphaFold2 or RoseTTAFold for initial structure prediction

  • Validate predictions through experimental approaches

  • Perform molecular dynamics simulations to assess stability

  • Identify potential functional sites through conservation mapping

The choice of method depends on protein characteristics, with NMR being advantageous for smaller proteins like CBU_0656 (110 amino acids), while X-ray crystallography provides higher resolution for well-diffracting crystals .

How can I design experiments to test if CBU_0656 functions as a bacterial effector protein?

To investigate whether CBU_0656 functions as a bacterial effector protein:

• Secretion System Analysis:

  • Assess presence of secretion signals using bioinformatics tools

  • Generate reporter fusions (CBU_0656-BlaM) to track translocation into host cells

  • Perform fractionation experiments to detect CBU_0656 in secreted fractions

  • Test dependency on known secretion systems through deletion mutants

• Host Cell Localization Studies:

  • Express fluorescently-tagged CBU_0656 in mammalian cells

  • Perform immunofluorescence microscopy to determine subcellular localization

  • Use cell fractionation followed by western blotting to confirm localization

  • Compare localization patterns with known C. burnetii effectors

• Functional Impact Assessment:

  • Identify cellular processes affected by CBU_0656 expression in host cells

  • Measure changes in host cell signaling pathways

  • Assess effects on cytoskeleton, vesicular trafficking, or cell death pathways

  • Compare phenotypes with known C. burnetii effector proteins

• Interaction Partner Identification:

  • Perform pull-down assays with host cell lysates

  • Identify binding partners using mass spectrometry

  • Validate key interactions with co-immunoprecipitation

  • Map interaction domains through truncation and point mutations

Recent studies have shown that many C. burnetii effectors target autophagy and proteasome pathways, which would be important to investigate for CBU_0656 .

What experimental design considerations are critical when studying the effects of CBU_0656 in infection models?

When designing experiments to study CBU_0656 in infection models:

• Experimental Design Parameters:

  • Use true experimental designs with appropriate controls:

    • Wild-type C. burnetii vs. CBU_0656 deletion mutants

    • Complemented mutants to confirm phenotype specificity

    • Multiple time points to capture temporal dynamics

    • Multiple cell types to assess tissue tropism

• Variable Control:

  • Standardize infection protocols (MOI, time of infection)

  • Control host cell conditions (passage number, confluence)

  • Use multiple technical and biological replicates

  • Include appropriate statistical analyses

• Readout Selection:

  • Bacterial replication (qPCR, colony counts)

  • Parasitophorous vacuole formation (immunofluorescence)

  • Host cell responses (cytokine production, gene expression)

  • Cell death and survival metrics

• Advanced Considerations:

  • Temporal profiling to determine when CBU_0656 functions during infection

  • Dose-response relationships using inducible expression systems

  • Comparison across different host species to assess conservation of function

  • Integration with systems biology approaches (proteomics, transcriptomics)

This experimental framework provides a rigorous approach to determine the role of CBU_0656 during C. burnetii infection while controlling for potential confounding variables and ensuring reproducibility .

How can I overcome solubility issues when expressing recombinant CBU_0656?

To address solubility challenges with recombinant CBU_0656:

• Expression Optimization Strategies:

  • Reduce expression temperature (16-20°C) to slow protein folding

  • Use weaker promoters or lower inducer concentrations

  • Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

  • Try auto-induction media for gradual protein expression

• Fusion Tag Approaches:

  • Test multiple solubility-enhancing tags:

    • MBP (maltose-binding protein)

    • SUMO

    • Thioredoxin

    • GST (glutathione S-transferase)

  • Compare with simple purification tags (His6, FLAG, Strep)

• Buffer Optimization:

  • Screen various buffer conditions:

    • pH range (6.0-9.0)

    • Salt concentrations (100-500 mM NaCl)

    • Additives (glycerol, arginine, detergents)

    • Reducing agents (DTT, TCEP, β-mercaptoethanol)

• Refolding Protocols:

  • Express as inclusion bodies if soluble expression fails

  • Develop refolding protocols using gradual dialysis

  • Test on-column refolding during purification

  • Validate proper folding using circular dichroism or fluorescence spectroscopy

The optimal approach will depend on the specific properties of CBU_0656, but a systematic screening of these conditions typically yields improvements in soluble protein expression.

What are the best approaches for studying protein-protein interactions involving CBU_0656?

For studying protein-protein interactions involving CBU_0656:

• In Vitro Methods:

  • Pull-down assays using tagged recombinant CBU_0656

  • Surface plasmon resonance for kinetic measurements

  • Isothermal titration calorimetry for thermodynamic parameters

  • ELISA-based binding assays for high-throughput screening

• Cell-Based Approaches:

  • Yeast two-hybrid screening against C. burnetii or host cell libraries

  • Mammalian two-hybrid for interactions in more native context

  • Split-reporter assays (luciferase, GFP complementation)

  • FRET/BRET for real-time interaction monitoring in live cells

• Proteomics Strategies:

  • Immunoprecipitation followed by mass spectrometry

  • BioID or APEX proximity labeling to capture transient interactions

  • Chemical cross-linking coupled with mass spectrometry

  • Label-free quantitative proteomics to measure interaction stoichiometry

• Validation Methods:

  • Co-immunoprecipitation of endogenous proteins

  • Co-localization studies using fluorescence microscopy

  • Functional assays based on predicted interaction outcomes

  • Mutational analysis to map interaction interfaces

Combining multiple complementary approaches provides the strongest evidence for genuine interactions and helps distinguish direct from indirect interactions.

How can I verify that my recombinant CBU_0656 protein retains its native structure and function?

To verify native structure and function of recombinant CBU_0656:

• Structural Integrity Assessment:

  • Circular dichroism spectroscopy to analyze secondary structure elements

  • Differential scanning fluorimetry to determine thermal stability

  • NMR HSQC experiments for tertiary structure fingerprinting

  • Size exclusion chromatography to confirm expected oligomeric state

• Functional Validation:

  • Develop activity assays based on predicted function from bioinformatics

  • Compare with native protein isolated from C. burnetii (if feasible)

  • Assess binding to predicted interaction partners

  • Evaluate ability to complement CBU_0656 knockout phenotypes

• Post-Translational Modification Analysis:

  • Mass spectrometry to identify modifications present in native vs. recombinant protein

  • Phosphoproteomic analysis if phosphorylation is suspected

  • Consider expression in eukaryotic systems if modifications are critical

  • Site-directed mutagenesis of putative modification sites

• Folding Assessment:

  • Limited proteolysis to probe for exposed flexible regions

  • Intrinsic fluorescence to assess tertiary structure around tryptophan residues

  • Antibody recognition if conformation-specific antibodies are available

  • Computational comparison with predicted structure models

The appropriate validation methods depend on the suspected function of CBU_0656, but structural analysis should always precede functional studies to ensure proper protein folding.