Recombinant Bacillus cereus UPF0747 protein BCE33L3678 (BCE33L3678), partial

What is the general classification and origin of Bacillus cereus?

Bacillus cereus is a Gram-positive, facultative anaerobic bacterium that belongs to the Bacillus cereus group. This organism is widely distributed in soil and various food products, with the potential to cause food poisoning through the production of enterotoxins and emetic toxins. B. cereus is closely related to other species in the Bacillus cereus group, including B. anthracis and B. thuringiensis, which can be differentiated through molecular typing methods such as Multilocus Sequence Typing (MLST) . The genetic diversity among B. cereus strains contributes to variations in virulence factors and protein expression profiles, including proteins like BCE33L3678.

What expression systems are recommended for producing recombinant B. cereus proteins?

For the expression of recombinant B. cereus proteins, including BCE33L3678, several systems can be employed:

• E. coli expression systems: BL21(DE3) strains are commonly used for initial expression trials due to their high yield and simplicity. For optimal expression:

  • Use autoinduction media to minimize toxicity

  • Optimize induction conditions (temperature: 16-37°C; IPTG concentration: 0.1-1.0 mM)

  • Consider fusion tags (His, GST, MBP) to enhance solubility

• Bacillus subtilis expression systems: When working with Bacillus proteins, B. subtilis can provide a more native-like environment with proper post-translational modifications.

• Cell-free expression systems: For potentially toxic proteins, cell-free systems can circumvent growth inhibition issues.

Expression should be verified through SDS-PAGE and Western blotting, with purification typically performed using affinity chromatography followed by size exclusion chromatography to ensure purity for downstream applications.

What are the optimal buffer conditions for maintaining BCE33L3678 stability?

The stability of recombinant BCE33L3678 protein requires careful consideration of buffer conditions:

For long-term storage, flash-freezing aliquots in liquid nitrogen and storing at -80°C with 10-15% glycerol is recommended to prevent freeze-thaw damage. Activity should be reassessed after storage to ensure functional integrity is maintained.

What purification protocols optimize yield and purity of recombinant BCE33L3678?

A multi-step purification strategy is recommended to achieve high purity recombinant BCE33L3678:

• Initial capture: If using His-tagged protein, employ immobilized metal affinity chromatography (IMAC) with Ni-NTA resin. Use low imidazole (10-20 mM) in washing buffers to minimize non-specific binding.

• Intermediate purification: Ion exchange chromatography based on the protein's theoretical pI (use anion exchange if pI < 7, cation exchange if pI > 7).

• Polishing: Size exclusion chromatography using a Superdex 75 or 200 column to separate monomeric protein from aggregates.

• Quality assessment: Analyze purity via SDS-PAGE (>95% purity) and verify identity through mass spectrometry.

For optimal results, maintain all buffers at 4°C and process samples quickly to minimize degradation. Consider adding stabilizers like 10% glycerol if the protein shows instability. Yield optimization may require adjusting cell lysis conditions (sonication parameters, detergent concentration for membrane-associated proteins).

How can researchers validate the biological activity of recombinant BCE33L3678?

Validating biological activity of BCE33L3678 requires multiple complementary approaches:

• Functional assays: Though specific enzymatic activity of UPF0747 family proteins is not well-characterized, general approaches include:

  • Substrate utilization assays if putative catalytic domains are identified

  • Protein-protein interaction studies using pull-down assays or surface plasmon resonance

  • Monitoring cellular phenotypes in BCE33L3678 knockout vs. complementation strains

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to confirm proper secondary structure

  • Thermal shift assays to evaluate protein stability

  • Dynamic light scattering to assess homogeneity and absence of aggregation

• In vitro transcription/translation systems:

  • Cell-free systems can be used to evaluate if BCE33L3678 affects protein synthesis

  • RNA binding assays if regulatory functions are suspected

For activity validation, a pH range assessment should be performed, as B. cereus proteins may have optimal activity under specific conditions. For instance, some B. cereus enzymes demonstrate optimal activity at acidic pH (3.5-5.0), similar to human Cathepsin D .

What methods are most effective for studying protein-protein interactions involving BCE33L3678?

To investigate protein-protein interactions involving BCE33L3678, several complementary techniques are recommended:

• Pull-down assays: Using tagged BCE33L3678 as bait to capture interacting partners from B. cereus lysate, followed by mass spectrometry identification. This approach can identify both strong and weak interactors.

• Bacterial two-hybrid systems: Modified for Gram-positive bacteria to identify direct protein interactions in a cellular context.

• Surface plasmon resonance (SPR): For quantitative measurement of binding kinetics and affinity constants between BCE33L3678 and putative binding partners.

• Cross-linking coupled with mass spectrometry: To capture transient interactions and identify interaction interfaces within protein complexes.

• Co-immunoprecipitation: If antibodies against BCE33L3678 are available, this method can confirm interactions under native conditions.

When performing these studies, including appropriate controls is essential to distinguish specific from non-specific interactions.

How might BCE33L3678 contribute to B. cereus virulence or pathogenicity?

While specific information about BCE33L3678's role in virulence is limited, we can analyze its potential contributions based on B. cereus pathogenicity mechanisms:

B. cereus pathogenicity primarily involves two types of toxins: diarrheal toxins (HBL, NHE, CytK) and emetic toxin (cereulide) . B. cereus strains commonly harbor multiple virulence genes, with 39% of isolates containing the hblACD gene cluster and 83% containing the nheABC gene cluster . The entFM gene is present in all B. cereus strains examined in recent studies, while cytK appears in 68% of isolates .

To investigate BCE33L3678's potential role in virulence:

• Comparative genomics approach: Analyze the presence and conservation of BCE33L3678 across virulent and non-virulent B. cereus strains, particularly those with well-characterized toxin profiles.

• Gene knockout studies: Generate BCE33L3678 deletion mutants and assess changes in:

  • Toxin production (quantify HBL, NHE, CytK, and cereulide levels)

  • Adherence to epithelial cells

  • Biofilm formation capacity

  • Resistance to environmental stressors

• Transcriptomic analysis: Compare gene expression patterns between wild-type and BCE33L3678 mutants under conditions that simulate the host environment (pH 5-7, 37°C, nutrient limitation).

• In vivo virulence models: Evaluate the impact of BCE33L3678 deletion on pathogenicity using appropriate animal or cell culture models.

Given that UPF0747 family proteins have unknown functions, BCE33L3678 could potentially function as a regulator of virulence factor expression, contribute to stress adaptation, or play a role in host cell interaction pathways.

What are the challenges in structural characterization of UPF0747 family proteins?

Structural characterization of UPF0747 family proteins, including BCE33L3678, presents several challenges:

• Expression and solubility issues:

  • UPF0747 proteins may form inclusion bodies when overexpressed

  • Optimization strategies include:

    • Reducing expression temperature (16-20°C)

    • Using solubility-enhancing fusion partners (MBP, SUMO)

    • Screening multiple construct boundaries to identify stable domains

• Crystallization barriers:

  • Proteins with unknown functions often lack known ligands that might stabilize conformation

  • Surface entropy reduction (replacing clusters of high-entropy residues with alanines) may improve crystal packing

  • High-throughput crystallization screening (>1000 conditions) is recommended

• NMR spectroscopy challenges:

  • Size limitations may require domain-by-domain analysis

  • Isotopic labeling (15N, 13C) is necessary for structural determination

  • Optimization of sample conditions (pH, ionic strength, temperature) is critical for spectral quality

• Computational prediction limitations:

  • Low sequence homology to characterized proteins limits template-based modeling

  • Ab initio methods may provide low-confidence models requiring experimental validation

  • Emerging approaches like AlphaFold2 may offer improved predictions

For BCE33L3678, a fragment-based approach targeting predicted domains may prove more successful than attempting to solve the structure of the full-length protein simultaneously.

How does protein BCE33L3678 expression vary across different strains of B. cereus?

B. cereus demonstrates considerable genetic diversity, with 368 isolates belonging to 192 different sequence types (STs) including 93 new STs as identified through multilocus sequence typing (MLST) . This genetic variability likely influences BCE33L3678 expression patterns across strains.

To investigate strain-specific BCE33L3678 expression:

• Comparative genomics analysis:

  • Examine BCE33L3678 gene presence, sequence conservation, and promoter regions across multiple B. cereus strains

  • Analyze genomic context to identify potential operon structures or regulatory elements

• Transcriptomic profiling:

  • Perform RT-qPCR or RNA-seq analysis of BCE33L3678 expression across representative strains from different clonal complexes

  • Compare expression under standardized growth conditions (rich media, minimal media, stress conditions)

• Protein level verification:

  • Develop specific antibodies against BCE33L3678 for Western blot analysis

  • Use targeted proteomics (PRM/MRM) for absolute quantification across strains

This comparative analysis may reveal correlations between BCE33L3678 expression levels and strain-specific characteristics such as virulence potential, ecological niche, or host association.

What approaches can be used to determine the biological function of UPF0747 proteins?

Determining the function of uncharacterized UPF0747 family proteins like BCE33L3678 requires an integrated approach:

• Bioinformatic analysis:

  • Sequence-based predictions: Search for conserved domains, motifs, or catalytic signatures

  • Structural homology modeling: Predict function based on structural similarity to characterized proteins

  • Genomic context analysis: Examine neighboring genes for functional clues (operon structure)

• Gene knockout and phenotypic screening:

  • Generate BCE33L3678 deletion mutants using CRISPR-Cas9 or allelic exchange

  • Perform high-throughput phenotypic screening:

    • Growth under various stress conditions (pH, temperature, oxidative stress)

    • Metabolic profiling using Biolog phenotype arrays

    • Antibiotic susceptibility testing (B. cereus strains show resistance to β-lactams and rifamycin)

• Protein interaction mapping:

  • Identify interaction partners using techniques described in section 2.3

  • Perform co-expression analysis to identify genes with correlated expression patterns

• Heterologous expression and complementation:

  • Express BCE33L3678 in model organisms with well-characterized genetics

  • Test if BCE33L3678 can complement known mutant phenotypes

• Biochemical function screening:

  • Test for enzymatic activities (hydrolase, transferase, etc.) using substrate panels

  • Examine protein modification states to identify potential regulatory mechanisms

By integrating data from these approaches, researchers can develop hypotheses about BCE33L3678 function that can be tested with targeted experiments.

How can CRISPR-Cas9 technology be optimized for studying BCE33L3678 in B. cereus?

CRISPR-Cas9 genome editing in B. cereus requires specific optimizations for studying BCE33L3678:

• Vector system selection:

  • Temperature-sensitive plasmids (pBCE or pHT01-based) for controllable replication

  • Inducible promoters (P​xyl or P​spac) to regulate Cas9 expression and minimize toxicity

• sgRNA design considerations:

  • Target specificity: Design sgRNAs with minimal off-target effects using tools like CHOPCHOP

  • PAM selection: Use NGG PAM sites near the target region

  • Efficiency prediction: Score potential sgRNAs using algorithms that predict cutting efficiency

• Homology-directed repair (HDR) template design:

  • For gene deletion: 500-1000 bp homology arms flanking BCE33L3678

  • For tagging: Insert tags while maintaining reading frame

  • For point mutations: Include silent mutations in the PAM or seed region to prevent re-cutting

• Transformation protocol optimization:

  • Prepare competent cells at early-mid logarithmic phase (OD600 0.3-0.5)

  • Use glycine (1-3%) in growth media to weaken the cell wall

  • Optimize electroporation parameters: field strength 20-25 kV/cm, resistance 200-400 Ω

• Screening strategies:

  • PCR-based genotyping to identify successful editing events

  • Sanger sequencing to confirm precise modifications

  • Phenotypic screening based on expected function

• Controls and validation:

  • Include complementation strains to verify phenotypes are due to BCE33L3678 modification

  • Use whole-genome sequencing to check for off-target effects

This optimized CRISPR-Cas9 approach enables precise genetic manipulation of BCE33L3678 to investigate its function through gene knockout, domain deletion, or targeted mutagenesis.

What is the potential role of BCE33L3678 in B. cereus stress response?

B. cereus must adapt to various environmental stressors in soil, food, and host environments. BCE33L3678 may contribute to stress adaptation through several potential mechanisms:

• Heat stress response:

  • Examine BCE33L3678 expression levels after heat shock (42-50°C)

  • Compare survival rates of wild-type vs. BCE33L3678 mutants under thermal stress

  • Investigate potential chaperone activity or interactions with known heat shock proteins

• Acid tolerance:

  • B. cereus must survive gastric transit (pH 2-3) to cause foodborne illness

  • Test BCE33L3678 mutants for survival at low pH conditions

  • Investigate if BCE33L3678 functions optimally in acidic conditions (pH 3.5-5.0), similar to some B. cereus enzymes

• Oxidative stress management:

  • Assess BCE33L3678 expression during exposure to hydrogen peroxide or superoxide

  • Measure oxidative damage markers in BCE33L3678 mutants

  • Test for potential antioxidant properties or interactions with redox-active proteins

• Nutrient limitation response:

  • Monitor BCE33L3678 expression during starvation conditions

  • Compare growth of wild-type and mutant strains in minimal media

  • Investigate potential roles in nutrient scavenging or metabolic adaptation

• Biofilm formation:

  • Evaluate BCE33L3678 contribution to biofilm development

  • Compare biofilm architecture and stability between wild-type and mutant strains

  • Investigate protein localization within biofilm structures

Stress response roles would be particularly relevant for B. cereus as a foodborne pathogen, as protein BCE33L3678 could potentially contribute to survival in food processing environments or during host infection.

What analytical techniques are most suitable for detecting post-translational modifications of BCE33L3678?

Post-translational modifications (PTMs) of BCE33L3678 can significantly impact its function and interactions. The following analytical techniques are recommended for comprehensive PTM characterization:

• Mass spectrometry-based approaches:

  • Liquid chromatography-tandem mass spectrometry (LC-MS/MS) using multiple fragmentation methods (CID, ETD, HCD) to preserve labile modifications

  • Top-down proteomics to analyze intact protein and identify PTM combinations

  • Targeted MS methods (PRM/MRM) for quantitative analysis of specific modifications

• Enrichment strategies for specific PTMs:

  • Phosphorylation: TiO2 or IMAC (Fe3+) enrichment

  • Glycosylation: Lectin affinity chromatography

  • Acetylation: Anti-acetyllysine antibody immunoprecipitation

• Site-specific modification analysis:

  • Site-directed mutagenesis of potential PTM sites

  • Expression in PTM-deficient strains to assess functional impact

• Specialized techniques for specific PTMs:

  • Phosphorylation: Phos-tag SDS-PAGE for mobility shift analysis

  • Glycosylation: Periodic acid-Schiff (PAS) staining

  • Disulfide bonds: Non-reducing vs. reducing SDS-PAGE

Sample preparation is critical for PTM analysis - rapid inactivation of cellular processes (flash freezing, TCA precipitation) helps preserve dynamic modifications. Different growth conditions should be tested, as PTMs often vary based on environmental factors.

How can researchers resolve discrepancies in experimental data related to BCE33L3678 function?

When faced with conflicting experimental results regarding BCE33L3678 function, a systematic troubleshooting approach is essential:

• Sources of experimental variability:

  • Strain differences: Genetic background can influence protein function

  • Growth conditions: Media composition, aeration, and growth phase affect protein expression

  • Protein preparation: Different purification methods may yield proteins with variable activity

• Validation strategies:

  • Independent methodologies: Confirm findings using orthogonal techniques

  • Inter-laboratory validation: Standardize protocols and reproduce experiments in different settings

  • Dose-response relationships: Test across a range of concentrations to identify threshold effects

• Statistical approaches:

  • Increase biological replicates (n≥3) to establish reproducibility

  • Apply appropriate statistical tests based on data distribution

  • Use power analysis to ensure sufficient sample size

• Controlling for confounding factors:

  • Generate multiple mutant strains using different strategies (deletion, point mutation)

  • Create complementation strains to verify phenotype restoration

  • Use closely related proteins as controls

• Resolution framework for contradictory results:

When publishing results, transparently report all experimental conditions and acknowledged limitations to facilitate reproducibility and resolution of discrepancies.

What considerations are important when designing BCE33L3678 for heterologous expression?

Designing BCE33L3678 constructs for optimal heterologous expression requires careful consideration of several factors:

• Sequence optimization:

  • Codon optimization based on the expression host (E. coli, B. subtilis, etc.)

  • Remove rare codons that may cause translational pausing

  • Optimize GC content for the expression system

  • Eliminate internal Shine-Dalgarno-like sequences that may cause translational problems

• Domain boundary selection:

  • Perform bioinformatic analysis to identify structured domains

  • Design multiple constructs with different N- and C-terminal boundaries

  • Consider structural information from homologous proteins when available

  • Include small extensions (±5 amino acids) around predicted domain boundaries

• Fusion tag selection:

  • N-terminal tags: His6, GST, MBP, SUMO for enhanced solubility

  • C-terminal tags: Consider when N-terminal structure is critical for function

  • Cleavage sites: Include protease recognition sequences (TEV, PreScission, thrombin)

  • Dual tags: Consider orthogonal purification strategies (His-MBP, His-FLAG)

• Expression vector selection:

  • Promoter strength: T7 for high expression, trc/tac for moderate expression

  • Induction system: IPTG, arabinose, tetracycline, or auto-induction

  • Copy number: Low copy plasmids for potentially toxic proteins

  • Selection marker: Consider antibiotic resistance compatible with host strain

• Design for structural studies:

  • Surface entropy reduction: Replace clusters of high-entropy residues (Lys, Glu) with alanines

  • Cysteine minimization: Replace non-essential cysteines to prevent non-native disulfide formation

  • Crystallization chaperones: Consider fusion to proteins like T4 lysozyme or MBP

Planning multiple construct strategies in parallel increases the likelihood of obtaining soluble, active protein for functional and structural studies.