Recombinant Bacillus cereus UPF0747 protein BCE_3965 (BCE_3965), partial

What is the basic identity and classification of UPF0747 protein BCE_3965?

UPF0747 protein BCE_3965 is an uncharacterized protein from Bacillus cereus strain ATCC 10987 / NRS 248. It belongs to the UPF0747 protein family, with UniProt accession number Q732E9, and is available as a partial recombinant protein . The protein is associated with the target name bshC, though its precise functional role remains to be fully characterized. The UPF designation indicates it belongs to a protein family whose function has not yet been experimentally determined, making it a potentially interesting target for basic research into B. cereus biology .

What are the optimal storage and handling conditions for BCE_3965?

The recombinant BCE_3965 protein has specific storage requirements to maintain stability and functionality. The lyophilized form maintains a shelf life of 12 months at -20°C/-80°C, while the liquid form has a reduced shelf life of approximately 6 months at the same temperatures . It is critical to avoid repeated freeze-thaw cycles as these can significantly compromise protein integrity. Working aliquots can be stored at 4°C for up to one week . For reconstitution, it is recommended to briefly centrifuge the vial before opening and to reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with the addition of 5-50% glycerol (final concentration) for long-term storage aliquots .

What expression systems are used for BCE_3965 production?

The recombinant BCE_3965 protein is produced in yeast expression systems, which provide appropriate eukaryotic post-translational modifications that may be important for protein folding and functionality . The protein achieves a purity of >85% as determined by SDS-PAGE analysis, making it suitable for most research applications . The specific tag type is determined during the manufacturing process and may vary between batches, which should be considered when designing experiments that might be affected by the presence of fusion tags .

How should researchers approach characterizing an uncharacterized protein like BCE_3965?

Characterization of an uncharacterized protein like BCE_3965 requires a multi-faceted approach. Begin with bioinformatic analysis to identify conserved domains, predict secondary structure, and search for orthologs in related species. Follow with experimental characterization including:

• Structural studies: X-ray crystallography, NMR spectroscopy, or cryo-electron microscopy

• Functional assays: Enzyme activity tests based on predicted functions

• Interaction studies: Pull-down assays, yeast two-hybrid screens, or protein microarrays

• Localization experiments: GFP fusion constructs or immunofluorescence

• Phenotypic analysis of knockout/knockdown mutants

Given the association with bshC target name, investigate potential roles in cellular processes related to this designation through targeted functional assays .

What strategies should be employed for BCE_3965 protein purification?

For optimal purification of recombinant BCE_3965, consider implementing a multi-step process tailored to its biochemical properties:

• Initial capture: Affinity chromatography utilizing the fusion tag present on the recombinant protein

• Intermediate purification: Ion exchange chromatography based on the protein's predicted isoelectric point

• Polishing: Size exclusion chromatography to separate aggregates and achieve high purity

The recombinant BCE_3965 product has a documented purity of >85% by SDS-PAGE, suggesting that industrial purification has already overcome major challenges . For research requiring higher purity, additional optimization may be necessary. Monitor protein stability throughout purification using dynamic light scattering and circular dichroism to ensure the protein maintains its native conformation.

How can researchers validate the functional activity of BCE_3965?

Validating the function of an uncharacterized protein presents unique challenges. Implement these methodological approaches:

• Comparative genomics: Analyze gene neighborhood and co-evolution patterns

• Transcriptomic analysis: Identify conditions that alter BCE_3965 expression

• Metabolomic profiling: Compare metabolite profiles between wild-type and BCE_3965 mutants

• Structure-guided hypotheses: Use solved or predicted structures to suggest potential functions

• Protein-protein interaction networks: Identify functional associations through interactome mapping

Document all experimental conditions meticulously, including protein concentration, buffer composition, temperature, and incubation times to ensure reproducibility. Validate findings using multiple experimental approaches to strengthen functional hypotheses .

How does BCE_3965 relate to B. cereus pathogenicity?

While direct evidence linking BCE_3965 to pathogenicity is limited, its investigation in the context of B. cereus virulence is warranted. B. cereus is known to cause food intoxication through various toxins and virulence factors, including nonhemolytic enterotoxin Nhe, hemolytic enterotoxin Hbl, enterotoxin FM, cytotoxin K (associated with diarrheal syndrome), and cereulide (associated with emetic syndrome) . To investigate potential roles of BCE_3965 in pathogenicity:

• Compare BCE_3965 expression levels between pathogenic and non-pathogenic strains

• Analyze co-expression patterns with known virulence factors

• Create knockout mutants and assess changes in virulence using in vitro and in vivo models

• Screen for interactions between BCE_3965 and known virulence factors

• Evaluate BCE_3965 contribution to stress responses that might enhance survival during infection

These approaches can reveal whether BCE_3965 contributes directly or indirectly to B. cereus pathogenicity .

How does BCE_3965 vary across different B. cereus strains?

B. cereus demonstrates significant genomic variability, with strains ranging from harmless to potentially lethal . Investigating BCE_3965 conservation and variation across different strains can provide insights into its evolutionary significance and potential specialized functions. Employ the following methodological approaches:

• Comparative genomic analysis: Align BCE_3965 sequences from multiple B. cereus strains to identify conserved regions and polymorphisms

• Phylogenetic analysis: Construct phylogenetic trees to understand evolutionary relationships

• Structure-function correlation: Map sequence variations to predicted structural features

• Expression analysis: Compare expression patterns of BCE_3965 across different strains under various conditions

• Functional conservation testing: Conduct complementation studies using BCE_3965 from different strains

This comparative approach may reveal whether BCE_3965 contributes to strain-specific characteristics or represents a core function conserved across the B. cereus group .

What molecular techniques are recommended for studying BCE_3965 interactions?

To elucidate the interaction network of BCE_3965, employ a combination of in vitro, in vivo, and in silico approaches:

• Affinity purification-mass spectrometry (AP-MS): Identify protein complexes containing BCE_3965

• Proximity-dependent biotin identification (BioID): Map the cellular neighborhood of BCE_3965

• Surface plasmon resonance (SPR): Determine binding kinetics of identified interactions

• Microscale thermophoresis (MST): Measure interactions under near-native conditions

• Förster resonance energy transfer (FRET): Visualize interactions in living cells

• Molecular docking and molecular dynamics simulations: Predict and validate structural basis of interactions

Validation through multiple complementary techniques is essential to distinguish genuine interactions from experimental artifacts. Control experiments should include tag-only controls and non-specific binding assessments .

How can researchers apply modern sequencing technologies to understand BCE_3965 function?

Next-generation sequencing techniques offer powerful approaches to functional characterization:

• RNA-Seq: Identify genes co-regulated with BCE_3965 under various conditions

• ChIP-Seq: If BCE_3965 has DNA-binding capabilities, map its genomic binding sites

• Ribo-Seq: Determine if BCE_3965 affects translation efficiency of specific transcripts

• CLIP-Seq: Identify any RNA interactions if BCE_3965 has RNA-binding potential

• Tn-Seq: Identify genetic interactions through transposon mutagenesis screens

• Whole-genome sequencing of evolved strains: Identify compensatory mutations in BCE_3965 mutants

Integrate these datasets using bioinformatic approaches to construct functional hypotheses that can be tested experimentally .

What structural analysis methods are most appropriate for BCE_3965?

Determining the structure of BCE_3965 is crucial for function prediction. Consider these methodological approaches:

• X-ray crystallography: Attempt crystallization trials using sparse matrix screens with various protein concentrations and precipitants

• NMR spectroscopy: For structural determination in solution, especially useful for identifying dynamic regions

• Cryo-electron microscopy: Particularly useful if BCE_3965 forms larger complexes

• Small-angle X-ray scattering (SAXS): For low-resolution shape information in solution

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): To identify flexible regions and binding interfaces

For each method, protein sample quality is paramount. Implement rigorous quality control using dynamic light scattering, circular dichroism, and thermal shift assays to ensure sample homogeneity before structural studies .

How can computational methods complement experimental structural studies of BCE_3965?

Computational approaches provide valuable structural insights when experimental data is limited:

• Homology modeling: Construct structural models based on related proteins with known structures

• Ab initio modeling: Predict structure from sequence alone using methods like AlphaFold2 or RoseTTAFold

• Molecular dynamics simulations: Explore conformational dynamics and stability

• Structure-based function prediction: Identify potential active sites or binding pockets

• Virtual screening: Predict potential ligands or substrates that might interact with BCE_3965

The integration of computational predictions with even limited experimental data can significantly accelerate structure determination and functional characterization .

What are the optimal methods for detecting BCE_3965 expression in different contexts?

Detection of BCE_3965 requires selecting appropriate methods based on research context:

• Western blotting: Using antibodies against BCE_3965 or its fusion tag for protein expression analysis

• Immunofluorescence: For cellular localization studies

• ELISA: For quantitative detection in complex samples

• Mass spectrometry: For unbiased detection and potential post-translational modification identification

• Real-time PCR: For transcript-level expression analysis

When developing detection methods, consider potential cross-reactivity with homologous proteins in the B. cereus group. Validation using BCE_3965 knockout controls is essential for confirming specificity .

How can researchers develop specific antibodies against BCE_3965?

Developing specific antibodies requires careful antigen design:

• Epitope prediction: Identify unique, surface-exposed regions of BCE_3965

• Peptide synthesis: Generate synthetic peptides corresponding to these regions

• Recombinant immunization: Use purified BCE_3965 for whole-protein immunization

• Hybridoma technology: For monoclonal antibody development

• Phage display: For selecting high-affinity antibody fragments

Rigorous validation of antibody specificity is crucial, especially given the potential for cross-reactivity with homologous proteins within the B. cereus group. Test antibodies against multiple strains and closely related species to ensure specificity .

How can gene editing techniques be applied to study BCE_3965 function?

Modern gene editing approaches offer powerful tools for functional characterization:

• CRISPR-Cas9 mutagenesis: Generate precise deletions or point mutations in BCE_3965

• Inducible expression systems: Control BCE_3965 expression levels temporally

• Complementation studies: Rescue mutant phenotypes with wild-type or modified BCE_3965

• Promoter reporter fusions: Monitor BCE_3965 expression under various conditions

• Domain swapping: Replace segments of BCE_3965 with corresponding regions from homologs

When designing gene editing experiments, consider potential polar effects on neighboring genes and implement appropriate controls, including complementation with the wild-type gene to confirm phenotypic specificity .

What transcriptomic approaches are useful for understanding BCE_3965 regulation?

Transcriptomic analyses provide insights into the regulation and functional context of BCE_3965:

• RNA-Seq: Compare global expression profiles between wild-type and BCE_3965 mutants

• Quantitative RT-PCR: For targeted analysis of specific gene expression changes

• Promoter analysis: Identify regulatory elements controlling BCE_3965 expression

• Transcription start site mapping: Determine the precise start site and potential alternative promoters

• Transcriptional regulator binding studies: Identify regulators controlling BCE_3965 expression

Integrating transcriptomic data with other genomic datasets can reveal co-regulated genes that may function in the same pathway as BCE_3965, providing clues to its biological role .

How does BCE_3965 research fit into broader B. cereus pathogenesis studies?

BCE_3965 research should be integrated with the broader context of B. cereus pathogenesis:

• Compare BCE_3965 expression during infection models versus laboratory conditions

• Investigate potential roles in stress responses relevant to host environments

• Assess BCE_3965 contribution to biofilm formation, a critical virulence factor

• Evaluate interactions with known virulence regulators

• Determine if BCE_3965 affects production or secretion of established toxins

This integration provides context for understanding the relative importance of BCE_3965 in B. cereus biology and pathogenesis .

What detection methods for B. cereus can be adapted to study BCE_3965 expression?

Established B. cereus detection methods can be modified to incorporate BCE_3965 analysis:

When adapting these methods, it's important to validate specificity and sensitivity for BCE_3965 detection in the context of complex samples .

What are common challenges in expressing and purifying BCE_3965, and how can they be addressed?

Researchers may encounter several challenges when working with BCE_3965:

• Protein solubility issues: Optimize expression conditions (temperature, induction timing) or use solubility tags

• Protein instability: Identify and implement stabilizing buffer conditions through thermal shift assays

• Low expression yields: Test different expression hosts or codon-optimized constructs

• Protein aggregation: Introduce additives like glycerol or low concentrations of detergents

• Proteolytic degradation: Include protease inhibitors and minimize handling time

A systematic approach to optimization, testing multiple conditions in parallel, can efficiently identify optimal parameters for BCE_3965 expression and purification .

How can researchers address conflicting or inconclusive results regarding BCE_3965 function?

When faced with conflicting data about BCE_3965 function:

• Verify reagent quality and experimental controls

• Test function under different physiological conditions that might affect protein activity

• Consider strain-specific differences that might influence results

• Employ multiple, complementary techniques to assess the same functional hypothesis

• Analyze potential post-translational modifications that might explain functional variability

• Consider context-dependent functions that might vary based on cellular environment

Transparent reporting of conflicting results contributes valuable information to the field and can lead to important discoveries about context-dependent protein functions .

What emerging technologies offer promise for BCE_3965 characterization?

Several cutting-edge technologies hold potential for advancing BCE_3965 research:

• Cryo-electron tomography: For visualizing BCE_3965 in its cellular context

• Single-molecule tracking: To monitor dynamics and interactions in living cells

• Microfluidics-based approaches: For high-throughput functional screening

• Synthetic biology: To test functional hypotheses through engineered systems

• Spatial transcriptomics: To map BCE_3965 expression in complex communities

• AlphaFold2 and related AI approaches: For improved structural predictions

Early adoption of these emerging technologies can provide competitive advantages in BCE_3965 research and lead to novel insights not accessible through conventional approaches .

How might BCE_3965 research contribute to broader understanding of uncharacterized protein families?

Research on BCE_3965 has implications beyond B. cereus biology:

• Development of generalizable approaches for UPF characterization

• Identification of novel protein functions potentially conserved across bacterial species

• Refinement of bioinformatic prediction algorithms through experimental validation

• Discovery of new antimicrobial targets if BCE_3965 serves essential functions

• Advancement of methods for integrating multi-omics data in protein function determination