Recombinant Bacillus cereus UPF0754 membrane protein BCAH820_0954 (BCAH820_0954)

What is Bacillus cereus UPF0754 membrane protein BCAH820_0954?

BCAH820_0954 is a membrane protein belonging to the UPF0754 family found in Bacillus cereus, a Gram-positive, rod-shaped bacterium commonly found in soil, food, and marine environments. The protein consists of 378 amino acids and is classified as a membrane protein based on its structural characteristics and hydrophobicity profile. The UPF0754 designation indicates that it belongs to a family of proteins with currently uncharacterized function (UPF = Uncharacterized Protein Family) . The protein has been assigned the UniProt ID B7JSD7, which serves as its unique identifier in protein databases .

What expression systems are recommended for recombinant BCAH820_0954 production?

For recombinant BCAH820_0954 production, Escherichia coli has been successfully employed as the expression host. The currently available recombinant protein includes an N-terminal His-tag to facilitate purification . When designing an expression strategy, researchers should consider:

• Codon optimization: The codon usage bias differs between B. cereus and expression hosts like E. coli, potentially affecting expression efficiency.

• Membrane protein expression challenges: As BCAH820_0954 is a membrane protein, expression may result in inclusion body formation, requiring optimization of conditions:

  • Reduced induction temperature (16-25°C)

  • Lower inducer concentrations

  • Use of specialized E. coli strains (e.g., C41(DE3), C43(DE3))

  • Co-expression with chaperones

• Fusion tags: While His-tags are commonly used, alternative fusion partners like MBP (maltose-binding protein) or SUMO might improve solubility and expression yields.

• Consideration of detergents: Early addition of mild detergents during cell lysis can improve extraction efficiency and prevent aggregation.

What purification strategies yield the highest purity and functional integrity of BCAH820_0954?

The current approach for BCAH820_0954 purification achieves greater than 90% purity as determined by SDS-PAGE . A comprehensive purification strategy should include:

• Initial capture: Immobilized metal affinity chromatography (IMAC) utilizing the His-tag

  • Ni-NTA or Co-based resins

  • Inclusion of low concentrations of imidazole (10-20 mM) in binding buffer to reduce non-specific binding

• Membrane protein solubilization:

  • Screen detergents (DDM, LDAO, Triton X-100) for optimal solubilization

  • Consider detergent concentration above critical micelle concentration (CMC)

• Secondary purification:

  • Size exclusion chromatography to remove aggregates and ensure homogeneity

  • Ion exchange chromatography as an orthogonal purification step

• Quality control assessments:

  • SDS-PAGE analysis

  • Western blotting

  • Dynamic light scattering for aggregation state

  • Circular dichroism for secondary structure confirmation

What are the optimal storage conditions for preserving BCAH820_0954 stability and activity?

Based on available information, the following storage recommendations apply to BCAH820_0954 :

• Short-term storage: Working aliquots can be stored at 4°C for up to one week.

• Long-term storage:

  • Store at -20°C/-80°C

  • Add glycerol to a final concentration of 5-50% (50% is recommended)

  • Prepare small aliquots to minimize freeze-thaw cycles

• Reconstitution protocol:

  • Briefly centrifuge the vial before opening

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

  • The standard storage buffer contains Tris/PBS with 6% trehalose at pH 8.0

How can researchers monitor and assess potential degradation of BCAH820_0954 over time?

Monitoring protein stability over time should involve multiple analytical approaches:

• SDS-PAGE analysis: Periodic assessment can reveal degradation products through the appearance of lower molecular weight bands.

• Size exclusion chromatography: Monitors aggregation state and can detect subtle changes in oligomerization or fragmentation.

• Activity assays: While the specific function of BCAH820_0954 remains uncharacterized, developing a functional assay would provide the most relevant stability indicator.

• Circular dichroism (CD): Provides information on secondary structure changes that might not be detectable by other methods.

• Differential scanning fluorimetry (DSF): Measures thermal stability (Tm) changes which often correlate with functional deterioration.

What experimental strategies can help elucidate the function of this uncharacterized membrane protein?

As a member of the UPF0754 family, the function of BCAH820_0954 remains to be fully characterized. Several complementary approaches can be employed:

• Bioinformatic analysis:

  • Sequence homology with characterized proteins

  • Identification of conserved domains using tools like PFAM, InterPro

  • Structural predictions using AlphaFold2 or similar tools

  • Genomic context analysis examining neighboring genes

• Gene knockout/knockdown studies:

  • CRISPR-Cas9 gene editing in B. cereus

  • Analysis of resultant phenotypes in various growth conditions

  • Transcriptomic analysis of knockout strains

• Protein-protein interaction studies:

  • Pull-down assays using the His-tagged protein

  • Bacterial two-hybrid screening

  • Crosslinking experiments followed by mass spectrometry

• Localization studies:

  • Fluorescent protein fusions

  • Immunofluorescence with antibodies against the protein or tag

  • Subcellular fractionation followed by Western blotting

• Functional reconstitution:

  • Incorporation into liposomes or nanodiscs

  • Transport assays with various substrates

  • Electrophysiological characterization if ion transport is suspected

How does BCAH820_0954 compare to similar membrane proteins in other Bacillus species?

BCAH820_0954 belongs to the Bacillus cereus group, which includes several closely related species including B. anthracis, B. thuringiensis, B. mycoides, B. pseudomycoides, and B. cytotoxicus . Comparative analysis reveals:

• Sequence conservation: Homologs exist across the B. cereus group with high sequence identity (>90%), suggesting conserved function.

• Structural features: Transmembrane topology predictions indicate similar membrane-spanning regions across homologs.

• Genomic context: Analysis of neighboring genes can provide functional clues if conserved across species.

• Expression patterns: RNA-seq data from different Bacillus species can indicate whether expression is constitutive or condition-specific.

• Evolutionary significance: BCAH820_0954 may represent a conserved adaptation specific to the B. cereus group lifestyle, potentially related to its ability to survive in diverse environments including soil, food, and occasionally as a human pathogen .

What role might BCAH820_0954 play in Bacillus cereus pathogenicity?

Bacillus cereus is known to cause foodborne illness and various infections including bacteremia, central nervous system infections, respiratory tract infections, and endophthalmitis . While BCAH820_0954's specific role in pathogenicity is not definitively established, several investigative approaches can explore this connection:

• Virulence correlation:

  • Compare expression levels between virulent and avirulent strains

  • Examine upregulation during infection models

  • Assess protein levels in clinical versus environmental isolates

• Host interaction studies:

  • Adhesion and invasion assays with host cells

  • Immune response stimulation measurements

  • Survival in phagocytes or serum resistance

• Animal model investigations:

  • Comparison of wild-type and BCAH820_0954 knockout strains in infection models

  • In vivo expression analysis during infection progression

  • Histopathological examination of infected tissues

The protein's membrane localization suggests potential roles in:

• Environmental sensing and adaptation

• Host cell interaction

• Resistance to host immune defenses

• Transport of nutrients or virulence factors

How can researchers effectively study the membrane topology and integration of BCAH820_0954?

Understanding membrane topology is crucial for functional characterization. Several complementary methods should be employed:

• Computational prediction:

  • Transmembrane helix prediction (TMHMM, Phobius)

  • Topology prediction algorithms (TOPCONS)

  • Signal peptide prediction (SignalP)

• Experimental verification methods:

  • Substituted cysteine accessibility method (SCAM): Systematically introduce cysteines and probe accessibility

  • PhoA/LacZ fusion analysis: Create fusion proteins at different positions to determine cytoplasmic versus periplasmic localization

  • Protease protection assays: Determine protein regions protected by the membrane

  • Fluorescence techniques: Förster resonance energy transfer (FRET) to measure distances between domains

• Structural biology approaches:

  • Cryo-electron microscopy of membrane-reconstituted protein

  • X-ray crystallography (challenging for membrane proteins)

  • NMR spectroscopy for dynamic regions

What strategies can overcome poor expression yields of recombinant BCAH820_0954?

Membrane proteins like BCAH820_0954 often present expression challenges. Systematic optimization approaches include:

• Expression system modifications:

  • Test multiple E. coli strains (BL21(DE3), C41(DE3), C43(DE3), Rosetta)

  • Consider alternative hosts (P. pastoris, insect cells)

  • Optimize codon usage for the expression host

  • Evaluate different promoter systems

• Construct design optimization:

  • Test truncated constructs removing potentially problematic regions

  • Evaluate different fusion partners (MBP, SUMO, TrxA)

  • Optimize tag placement (N-terminal vs. C-terminal)

  • Consider dual tagging strategies for verification

• Culture condition modifications:

  • Reduce temperature after induction (16-25°C)

  • Test various induction OD values

  • Vary inducer concentration

  • Extend expression time (24-72 hours)

  • Supplement with ligands or stabilizing agents

• Solubilization improvements:

  • Systematic detergent screening

  • Addition of lipids during solubilization

  • Test various buffer compositions (pH, salt concentration)

How can researchers distinguish between native function and artifacts when studying BCAH820_0954?

When characterizing proteins of unknown function, distinguishing genuine activity from experimental artifacts requires rigorous controls:

• Negative control proteins:

  • Inactive point mutants (targeting predicted functional residues)

  • Unrelated membrane proteins expressed and purified identically

  • Heat-denatured BCAH820_0954

• Activity verification approaches:

  • Concentration-dependent effects

  • Substrate specificity analysis

  • Inhibitor studies if applicable

  • Correlation with structural changes

• In vivo validation:

  • Complementation of knockout phenotypes

  • Structure-function correlation studies

  • Physiological relevance assessment

• Technical considerations:

  • Tag interference assessment (comparing tagged vs. untagged proteins)

  • Detergent effect control experiments

  • Reproducibility across different purification batches