Recombinant Bacillus cereus UPF0754 membrane protein BCAH187_A1042 (BCAH187_A1042)

What are the optimal storage and handling conditions for recombinant BCAH187_A1042 protein?

For optimal stability and activity retention, the recombinant BCAH187_A1042 protein should be handled according to these research-validated protocols:

• Storage conditions: Store at -20°C/-80°C upon receipt, with -80°C recommended for long-term storage .

• Aliquoting strategy: Divide into single-use aliquots to avoid repeated freeze-thaw cycles, which significantly reduce protein activity .

• Working temperature: For short-term use (up to one week), working aliquots can be maintained at 4°C .

• Buffer composition: The protein is typically stored in Tris/PBS-based buffer with 6% Trehalose, pH 8.0 .

• Reconstitution method:

  • Centrifuge vial briefly before opening

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

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

  • Aliquot for long-term storage

The addition of glycerol as a cryoprotectant is particularly important for maintaining protein stability during freeze-thaw cycles when working with membrane proteins like BCAH187_A1042.

What expression systems are optimal for recombinant BCAH187_A1042 production?

The established method for BCAH187_A1042 expression utilizes E. coli as the host organism with the following methodological considerations:

• Expression host: E. coli has been validated for successful expression of BCAH187_A1042 with an N-terminal His-tag . This system balances yield with proper folding of the membrane protein.

• Enhancement strategy: Co-expression with B. cereus phospholipase C (PLC) can significantly increase extracellular production of recombinant proteins in E. coli . This approach:

  • Enhances membrane permeability without causing obvious cell lysis

  • Facilitates protein release into the culture medium

  • Is especially effective for proteins with lower molecular mass (BCAH187_A1042 at 378 amino acids qualifies as moderate size)

• Scale-up considerations: The PLC co-expression strategy has been successfully scaled up in 3-L fermentors without problematic foam formation, making it suitable for larger-scale research applications .

• Alternative considerations: For researchers requiring protein with native conformation, expression in a Bacillus-based system might provide a more native-like membrane environment, though this typically results in lower yields than E. coli systems.

What purification strategies maximize yield and purity of BCAH187_A1042?

An optimized purification protocol for BCAH187_A1042 would incorporate these methodological approaches:

• Affinity chromatography: The N-terminal His-tag enables efficient purification using Ni-NTA or similar metal affinity resins . Optimization of imidazole concentrations in wash and elution buffers is critical for membrane proteins.

• Membrane protein solubilization:

  • Use mild detergents like n-dodecyl-β-D-maltoside (DDM) or CHAPS at concentrations above their critical micelle concentration

  • Test detergent screening panels to identify optimal solubilization conditions

  • Include stabilizing agents like glycerol (10-20%) in extraction buffers

• Size exclusion chromatography: As a polishing step to remove aggregates and achieve >90% purity as verified by SDS-PAGE .

• Yield optimization metrics:

  Optimization Parameter	Typical Range	Notes
  Induction temperature	16-30°C	Lower temperatures favor proper folding
  Induction duration	4-18 hours	Membrane proteins often require extended expression
  IPTG concentration	0.1-1.0 mM	Lower concentrations may improve folding
  Cell density at induction	OD600 0.6-0.8	Critical for balancing growth and expression

Researchers should conduct small-scale optimization experiments to determine the specific conditions that maximize yield for their particular expression construct and E. coli strain.

How can CRISPR/Cas9 technology be applied to study BCAH187_A1042 in Bacillus cereus?

CRISPR/Cas9 technology offers a powerful approach for genomic manipulation of BCAH187_A1042 in B. cereus with these methodological considerations:

• System components: A validated CRISPR/Cas9 system for B. cereus includes:

  • A plasmid expressing Cas9 protein under inducible control (mannose-inducible promoter has been validated)

  • A guide RNA targeting the BCAH187_A1042 gene

  • Homology-directed repair templates for precise modifications

• Transformation protocol:

  • Prepare electrocompetent B. cereus cells

  • Mix ~1 μg plasmid DNA with competent cells

  • Electroporate (0.6 kV, 500 Ω, 25 μF) in a 0.1 cm gap cuvette

  • Recover cells in LB medium for 1 hour at 30°C with shaking

  • Select transformants on kanamycin (25 μg/ml) containing plates

• Cas9 induction protocol:

  • Culture verified colonies in liquid medium with kanamycin for 3 hours at 30°C

  • Add mannose (final concentration 0.4%, w/v) to induce Cas9 expression

  • Incubate for 13 hours at 28°C

  • Transfer cultures to fresh medium (1% dilution) and repeat induction

• Verification methods:

  • PCR amplification of the target region using primers flanking the modification site

  • Sequencing to confirm precise modifications

  • Phenotypic assays to validate functional consequences

This methodology has demonstrated high efficiency in B. cereus, with reported success rates of 20-100% for various genomic targets, making it suitable for studying BCAH187_A1042 function through precise genetic modifications .

What experimental approaches can determine the function of UPF0754 membrane proteins like BCAH187_A1042?

A comprehensive functional characterization of BCAH187_A1042 would employ these methodological approaches:

• Genetic knockout studies:

  • Create a BCAH187_A1042 deletion mutant using CRISPR/Cas9

  • Conduct comparative phenotypic analysis between wild-type and mutant strains

  • Examine growth rates, stress responses, membrane integrity, and virulence phenotypes

• Protein interaction analysis:

  • Perform pull-down assays using His-tagged BCAH187_A1042

  • Conduct bacterial two-hybrid screening

  • Analyze the protein interactome using mass spectrometry

• Localization studies:

  • Generate fluorescent protein fusions to determine subcellular localization

  • Perform immunogold electron microscopy with anti-His antibodies

  • Conduct fractionation studies to confirm membrane association

• Structure-function analysis:

  • Generate point mutations in conserved domains

  • Create chimeric proteins with homologous UPF0754 family members

  • Assess functional complementation between species

• Transcriptional analysis:

  • Determine expression patterns under various growth conditions

  • Identify transcriptional regulators controlling BCAH187_A1042 expression

  • Conduct RNA-seq to identify genes co-regulated with BCAH187_A1042

The combination of these approaches would provide a comprehensive understanding of BCAH187_A1042's biological role in B. cereus membrane biology and cellular processes.

How does co-expression with phospholipase C enhance recombinant protein production, and is it applicable to BCAH187_A1042?

Co-expression with B. cereus phospholipase C (PLC) represents an innovative approach for enhancing extracellular production of recombinant proteins with specific mechanistic advantages:

• Mechanism of action:

  • PLC catalyzes the hydrolysis of phospholipids to produce sn1,2-diacylglycerides and organic phosphate

  • This increases membrane permeability without causing obvious cell lysis

  • Enhanced permeability facilitates protein secretion into the culture medium

• Efficiency factors:

  • Enhancement correlates strongly with molecular mass of the target protein

  • Proteins with lower molecular mass show greater enhancement

  • For BCAH187_A1042 (~42 kDa), significant enhancement would be expected

• Performance metrics:

  Protein Type	Molecular Mass	Fold Increase in Extracellular Production
  Secretory enzymes	~43 kDa	4.0-fold
  Cytosolic enzymes	~51 kDa	Up to 88.3% of total activity in supernatant

• Methodology for implementation:

  • Design co-expression vector containing both BCAH187_A1042 and PLC genes

  • Express PLC without its signal peptide to enhance extracellular localization

  • Optimize induction conditions for both proteins

  • Harvest directly from culture supernatant for simplified purification

• Scale-up advantages:

  • Unlike other permeabilization methods, PLC co-expression doesn't generate problematic foam

  • Successfully tested in 3-L fermentors for scaled production

  • Provides a controlled fermentation process suitable for larger research applications

This strategy would be particularly beneficial for researchers seeking higher yields of BCAH187_A1042 for structural or functional studies, with the added advantage of simplified downstream purification from the culture supernatant rather than cell lysates.

What analytical methods are most appropriate for assessing the structural integrity of recombinant BCAH187_A1042?

A comprehensive structural analysis of BCAH187_A1042 requires multiple complementary techniques:

• Primary integrity assessment:

  • SDS-PAGE to verify molecular weight and initial purity

  • Western blot with anti-His antibodies to confirm identity

  • Mass spectrometry to verify exact mass and detect post-translational modifications

• Secondary structure analysis:

  • Circular dichroism (CD) spectroscopy in the far-UV range (190-250 nm)

  • Fourier-transform infrared spectroscopy (FTIR) with attenuated total reflection

  • These methods can estimate α-helical content, which is typically high in membrane proteins

• Tertiary structure examination:

  • Intrinsic tryptophan fluorescence spectroscopy

  • Differential scanning calorimetry to assess thermal stability

  • Limited proteolysis combined with mass spectrometry to identify stable domains

• Membrane integration analysis:

  • Detergent resistance assays to assess membrane association strength

  • Fluorescence-based membrane reconstitution assays

  • Cryo-electron microscopy of membrane-reconstituted protein

• Aggregation monitoring:

  • Dynamic light scattering to assess homogeneity

  • Size-exclusion chromatography to detect oligomeric states

  • Analytical ultracentrifugation for detailed quaternary structure analysis

These analytical approaches provide complementary data to verify that the recombinant BCAH187_A1042 maintains its native structural characteristics and is suitable for downstream functional or structural biology applications.

How does BCAH187_A1042 compare to homologous UPF0754 proteins in other bacterial species?

A systematic comparative analysis of BCAH187_A1042 with homologous UPF0754 family proteins reveals evolutionary patterns and functional implications:

• Phylogenetic distribution:

  • UPF0754 family proteins are found across multiple Bacillus species

  • Homologs exist in related Gram-positive bacteria with varying sequence conservation

  • Sequence conservation is typically higher in the predicted transmembrane regions

• Sequence conservation analysis:

  • Conserved motifs include membrane-spanning helices and potential functional domains

  • The N-terminal region (residues 1-50) shows high conservation, suggesting functional importance

  • The C-terminal region exhibits greater variability between species

• Structural predictions:

  • Bioinformatic analysis predicts 7-9 transmembrane helices in most UPF0754 family members

  • Topology models suggest the N-terminus is located in the cytoplasm

  • Conserved charged residues in transmembrane regions may indicate ion transport or sensing functions

• Genomic context:

  • In many Bacillus species, UPF0754 genes cluster with genes involved in membrane homeostasis

  • Operonic arrangements differ between pathogenic and non-pathogenic species

  • This contextual information provides clues to potential functional roles

• Expression patterns:

  • Transcriptomic data indicates differential regulation between species

  • In B. cereus, expression may correlate with growth phase or stress conditions

  • Comparative expression studies could reveal functional conservation or divergence

This comparative framework provides researchers with evolutionary context for BCAH187_A1042 and may guide functional hypotheses based on better-characterized homologs in other bacterial species.

What potential biotechnological applications exist for BCAH187_A1042 and related membrane proteins?

BCAH187_A1042 and related UPF0754 membrane proteins have several potential biotechnological applications that researchers could explore:

• Membrane protein engineering platform:

  • The established expression and purification protocols for BCAH187_A1042 provide a template for engineering novel membrane proteins

  • The protein's structure could be used as a scaffold for designing membrane-spanning biotechnological tools

• Biosensor development:

  • If the protein functions in ion transport or sensing, it could be engineered as a biosensor component

  • Fusion with reporter proteins could create membrane-anchored sensing systems

• Vaccine development:

  • The CRISPR/Cas9 system developed for B. cereus could be used to generate attenuated strains through BCAH187_A1042 modification

  • These strains could serve as live attenuated vaccine candidates or delivery vehicles

• Protein production enhancement:

  • Insights from the phospholipase C co-expression system could be applied to improve production of other challenging membrane proteins

  • The methodology developed for BCAH187_A1042 expression contributes to the broader field of membrane protein production

• Structural biology research:

  • As a representative of the UPF0754 family, structural determination of BCAH187_A1042 would contribute to understanding membrane protein folding and stability

  • Crystallization or cryo-EM studies could reveal novel structural motifs with broader implications

These applications highlight how fundamental research on BCAH187_A1042 can translate into biotechnological innovations, particularly in areas related to bacterial membrane biology and protein engineering.

What are the most promising research questions regarding BCAH187_A1042 function?

Several high-priority research questions warrant investigation to advance understanding of BCAH187_A1042:

• Functional role determination:

  • Does BCAH187_A1042 function in transport, signaling, or structural membrane organization?

  • How does deletion affect B. cereus survival under various stress conditions?

  • Is the protein essential under specific environmental conditions?

• Protein-protein interaction networks:

  • What proteins directly interact with BCAH187_A1042 in the membrane?

  • Does it form homo-oligomeric structures or heterocomplexes?

  • How do these interactions change under different physiological conditions?

• Structural characterization:

  • What is the high-resolution structure of BCAH187_A1042?

  • How does the structure relate to its membrane topology and function?

  • What structural features are conserved across the UPF0754 family?

• Regulation of expression:

  • What environmental signals regulate BCAH187_A1042 expression?

  • Which transcription factors control its expression?

  • How does its expression pattern correlate with B. cereus pathogenicity?

• Comparative analysis across Bacillus species:

  • How does function vary between homologs in pathogenic vs. non-pathogenic Bacillus species?

  • Can functional differences be mapped to specific sequence variations?

  • Does horizontal gene transfer play a role in UPF0754 family evolution?

Addressing these questions through systematic research approaches would significantly advance understanding of this understudied membrane protein family and potentially reveal new insights into bacterial membrane biology.

How can emerging technologies advance the study of BCAH187_A1042?

Emerging technologies offer new opportunities for comprehensive characterization of BCAH187_A1042:

• Cryo-electron microscopy advances:

  • Recent advances in single-particle cryo-EM enable high-resolution structure determination of membrane proteins

  • Application to BCAH187_A1042 could resolve its structure without crystallization

  • Complementary techniques like cryo-electron tomography could visualize the protein in its native membrane context

• Integrative structural biology approaches:

  • Combining computational modeling with experimental constraints from cross-linking mass spectrometry

  • Hydrogen-deuterium exchange mass spectrometry to map dynamics and interaction surfaces

  • Deep learning-based structure prediction methods like AlphaFold2 for model generation

• Advanced genetic tools:

  • Base editing technologies for precise modification without double-strand breaks

  • CRISPRi/CRISPRa for regulated expression modulation without permanent genetic changes

  • Multiplexed CRISPR systems for simultaneous modification of BCAH187_A1042 and potential interaction partners

• Single-cell technologies:

  • Single-cell RNA-seq to examine expression heterogeneity within bacterial populations

  • Time-lapse microscopy with fluorescent protein fusions to track dynamic localization

  • Microfluidic approaches to correlate expression with single-cell phenotypes

• Advanced computational approaches:

  • Molecular dynamics simulations of BCAH187_A1042 in lipid bilayers

  • Systems biology modeling to integrate BCAH187_A1042 into cellular networks

  • Evolutionary coupling analysis to predict functionally important residues

These emerging technologies would provide unprecedented insights into BCAH187_A1042 structure, function, and biological context, potentially revealing unexpected roles in bacterial physiology.