Recombinant Bacillus cereus UPF0398 protein BCE_1688 (BCE_1688)

What is the BCE_1688 protein and what organism does it originate from?

BCE_1688 is an uncharacterized protein (UPF0398 family) encoded by the BCE_1688 gene in Bacillus cereus strain ATCC 10987 / NRS 248. The protein belongs to the UPF0398 family of proteins with currently unknown function. As a recombinant protein, it is typically expressed in mammalian cell expression systems to maintain proper folding and post-translational modifications . While its exact biological role remains to be fully elucidated, its presence in the virulent B. cereus suggests potential relevance to bacterial physiology or pathogenicity mechanisms. B. cereus is known for producing various virulence factors regulated by pleiotropic regulators like PlcR, which controls multiple genes encoding degradative enzymes and enterotoxins .

How does BCE_1688 relate to other proteins in Bacillus cereus virulence mechanisms?

While direct evidence linking BCE_1688 to B. cereus virulence mechanisms is limited in the current literature, it's important to consider it within the broader context of B. cereus pathogenicity. B. cereus produces several virulence factors, including phospholipases, enterotoxins (like hemolysin BL and non-hemolytic enterotoxin), and proteases, many of which are regulated by PlcR, a pleiotropic regulator that binds to conserved palindromic DNA sequences .

Unlike well-characterized virulence factors such as hemolysin BL (HBL), which has been shown to activate the NLRP3 inflammasome and induce inflammatory responses in host cells, BCE_1688's specific role remains undetermined . Research exploring potential interactions between BCE_1688 and known virulence pathways could provide valuable insights into whether this protein contributes to B. cereus pathogenicity or serves other metabolic or cellular functions.

What are the optimal storage and handling conditions for recombinant BCE_1688 protein?

For optimal stability and activity retention, BCE_1688 recombinant protein requires specific storage conditions. The shelf life of the liquid form is generally 6 months when stored at -20°C/-80°C, while the lyophilized form maintains stability for approximately 12 months at the same temperature range . Repeated freeze-thaw cycles significantly reduce protein integrity and should be avoided; instead, prepare smaller working aliquots for routine use.

For short-term storage of working aliquots, maintain at 4°C for no longer than one week. When handling the protein, minimize exposure to room temperature and avoid contamination. Before opening the vial, a brief centrifugation is recommended to bring contents to the bottom. For reconstitution of lyophilized protein, use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL, and consider adding glycerol to a final concentration of 5-50% (with 50% being the standard recommendation) before aliquoting for long-term storage . These careful handling procedures help preserve the structural integrity and functional properties of the protein for experimental applications.

What is the recommended reconstitution protocol for lyophilized BCE_1688 protein?

The reconstitution protocol for lyophilized BCE_1688 protein requires a methodical approach to ensure optimal protein recovery and activity. Begin by centrifuging the vial briefly (30 seconds at low speed) to collect the lyophilized material at the bottom. Reconstitute the protein in deionized sterile water to achieve a concentration between 0.1-1.0 mg/mL, allowing the solution to stand for 5-10 minutes at room temperature with occasional gentle mixing .

For long-term storage stability, add glycerol to a final concentration of 5-50%, with 50% being the standard recommendation for maximal protection against freeze-damage. After reconstitution, the solution should be divided into small working aliquots (typically 10-20 μL) in sterile microcentrifuge tubes and stored at -20°C or preferably -80°C. This aliquoting strategy prevents protein degradation from repeated freeze-thaw cycles . When thawing an aliquot for experimental use, warm it gently to room temperature with minimal agitation, and keep it on ice during the experiment to maintain protein integrity.

What expression systems are most effective for producing recombinant BCE_1688 protein?

Mammalian cell expression systems have proven most effective for the production of recombinant BCE_1688 protein with high purity and proper folding . This approach offers several advantages over prokaryotic systems, particularly for proteins that may require specific post-translational modifications or proper disulfide bond formation. The mammalian system helps ensure that the recombinant BCE_1688 protein maintains its native conformation and biological activities.

When designing expression protocols, researchers should consider optimizing the following parameters:

• Expression vector selection (with appropriate promoters and selection markers)

• Cell line selection (HEK293, CHO, or other mammalian lines)

• Transfection efficiency optimization

• Culture conditions (temperature, media composition, and induction timing)

• Purification strategy (typically involving affinity chromatography)

For analytical purposes, the expressed protein can be validated using SDS-PAGE, where BCE_1688 typically demonstrates >85% purity . Western blotting with specific antibodies can confirm protein identity, while mass spectrometry provides precise molecular weight determination and potential identification of post-translational modifications that might be functionally significant.

What experimental approaches are recommended for characterizing the function of BCE_1688?

Characterizing the function of BCE_1688 requires a multi-faceted approach combining bioinformatic, biochemical, and cellular methods. Begin with comparative sequence analysis against known protein domains and structures using tools like BLAST, PFAM, and AlphaFold to generate functional hypotheses. Follow with recombinant protein expression and purification as described earlier, ensuring >85% purity as verified by SDS-PAGE .

For biochemical characterization, conduct enzymatic assays based on predicted functions, assessing various substrates and reaction conditions. Structural studies using X-ray crystallography or NMR spectroscopy can provide insights into functional domains and potential interaction surfaces. For cellular studies, examine BCE_1688's potential role in B. cereus by creating gene knockout strains and assessing phenotypic changes in growth, stress response, or virulence.

Protein-protein interaction studies using pull-down assays, co-immunoprecipitation, or yeast two-hybrid screens can identify binding partners, potentially linking BCE_1688 to known bacterial pathways. Additionally, transcriptomic analysis comparing wild-type and BCE_1688-deficient strains can reveal affected pathways. Given that B. cereus produces virulence factors regulated by PlcR , investigating potential relationships between BCE_1688 and the PlcR regulon could provide valuable functional insights into this uncharacterized protein.

How can researchers assess potential interactions between BCE_1688 and host immune responses?

To investigate potential interactions between BCE_1688 and host immune responses, researchers should employ a systematic approach that examines various aspects of innate and adaptive immunity. Initial studies should focus on determining whether purified BCE_1688 elicits inflammatory responses in immune cell models, such as macrophages or dendritic cells, by measuring cytokine production (TNF, IL-1β, IL-18) and activation markers.

Drawing from methodologies used to study other B. cereus toxins, researchers can assess whether BCE_1688 activates inflammasome pathways, particularly the NLRP3 inflammasome which is known to respond to various B. cereus components . This would involve comparing responses in wild-type cells versus cells deficient in key inflammasome components (NLRP3, ASC, caspase-1) or using inflammasome inhibitors like MCC950.

Additional approaches should include:

• Cell-based assays to detect membrane interactions, pore formation, or cellular entry

• Microscopy techniques to track protein localization within host cells

• Receptor binding studies to identify potential host targets

• Signaling pathway analysis (NF-κB, MAPK) to determine downstream effects

• Comparative studies with known B. cereus virulence factors like hemolysin BL

These approaches could reveal whether BCE_1688 contributes to pathogenesis through immunomodulatory effects or represents a potential target for host immune recognition.

What techniques are most informative for studying the genetic regulation of BCE_1688 expression?

Understanding the genetic regulation of BCE_1688 expression requires a comprehensive approach combining molecular genetics and systems biology techniques. Begin with promoter analysis using bioinformatic tools to identify potential regulatory elements, particularly checking for conserved palindromic sequences that might serve as binding sites for pleiotropic regulators like PlcR, known to control numerous virulence-associated genes in B. cereus .

Reporter gene assays using the BCE_1688 promoter region fused to reporter genes (such as luciferase or GFP) can quantitatively measure promoter activity under various growth conditions, revealing environmental triggers for expression. Chromatin immunoprecipitation (ChIP) experiments can identify transcription factors that directly bind to the BCE_1688 promoter region in vivo.

RNA-seq analysis comparing transcript levels across different growth phases, stress conditions, and nutrient environments can establish the expression profile of BCE_1688 in relation to other genes. Quantitative RT-PCR provides precise measurement of BCE_1688 mRNA levels for specific condition testing. For a systems-level understanding, construct regulatory network models incorporating BCE_1688 with other genes responding to similar conditions or regulators.

Since B. cereus virulence genes often show growth phase-dependent expression patterns with PlcR activation during stationary phase , examining BCE_1688 expression across growth phases could be particularly informative for understanding its potential role in bacterial physiology or virulence.

How might researchers explore potential roles of BCE_1688 in Bacillus cereus pathogenicity mechanisms?

Investigating BCE_1688's potential role in B. cereus pathogenicity requires a comprehensive experimental approach. Begin by generating a clean deletion mutant (ΔBCE_1688) and corresponding complemented strain to establish causality in observed phenotypes. Compare the ΔBCE_1688 mutant with the parental strain in various infection models, including mammalian cell culture systems (epithelial cells, macrophages) and invertebrate models (Galleria mellonella, Caenorhabditis elegans), measuring virulence parameters like cytotoxicity, bacterial persistence, and host survival.

Conduct transcriptomic and proteomic analyses comparing the mutant and wild-type strains during infection to identify pathways affected by BCE_1688 deletion. Specifically examine whether BCE_1688 influences the expression or activity of established virulence factors like hemolysin BL (HBL), which is known to activate the NLRP3 inflammasome . Investigate whether BCE_1688 contributes to specific virulence mechanisms such as host cell invasion, immune evasion, or toxin production.

Given that PlcR regulates numerous virulence factors in B. cereus , determine if BCE_1688 is part of the PlcR regulon by comparing its expression in wild-type and PlcR-deficient backgrounds. Additionally, assess whether BCE_1688 interacts with other regulatory networks controlling virulence gene expression in B. cereus. These systematic approaches would provide comprehensive insights into whether BCE_1688 contributes to B. cereus pathogenicity and the mechanisms involved.

What are the challenges in studying BCE_1688 and how can researchers address contradictory experimental findings?

Studying an uncharacterized protein like BCE_1688 presents several significant challenges that researchers should anticipate and address methodically. The primary challenge is the lack of established functional information, which necessitates multiple parallel hypotheses to be tested simultaneously. When contradictory findings emerge across different experimental systems or laboratories, researchers should implement a systematic troubleshooting approach.

First, examine methodological differences that could explain contradictions, including:

• Protein preparation variations (expression systems, purification methods, storage conditions)

• Experimental conditions (buffer compositions, temperature, pH, presence of cofactors)

• Cell or animal models used (different strains, genetic backgrounds, culture conditions)

• Detection methods and their sensitivity thresholds

For resolving contradictions, implement the following strategies:

• Direct replication studies with identical protocols across laboratories

• Orthogonal experimental approaches to test the same hypothesis

• Dose-response studies to identify threshold effects

• Time-course experiments to capture temporal dynamics

• Collaborative cross-validation between research groups

Additionally, consider strain-specific effects, as B. cereus represents a diverse group of bacteria with strain-to-strain variation. The specific genetic background of strain ATCC 10987 / NRS 248 (the source of BCE_1688) may influence protein function in ways not generalizable to other B. cereus strains . Finally, maintain transparency in reporting both positive and negative results, including specific experimental conditions, to facilitate accurate interpretation of seemingly contradictory findings.

How can computational approaches enhance our understanding of BCE_1688 structure-function relationships?

Computational approaches offer powerful complementary tools for elucidating BCE_1688 structure-function relationships, particularly valuable given the experimental challenges associated with uncharacterized proteins. Begin with advanced sequence analysis methods including position-specific scoring matrices, hidden Markov models, and deep learning approaches to detect remote homologies that might not be evident through standard BLAST searches.

Employ state-of-the-art protein structure prediction tools like AlphaFold2 or RoseTTAFold to generate high-confidence structural models of BCE_1688. These models can reveal potential functional sites and domains not apparent from sequence analysis alone. Follow with molecular dynamics simulations to study the protein's conformational flexibility and stability under various conditions, providing insights into potential functional mechanisms.

For functional prediction, implement:

• Binding site prediction algorithms to identify potential interaction surfaces

• Virtual screening against libraries of small molecules or peptides to predict binding partners

• Protein-protein docking simulations with potential interactors from the B. cereus proteome

• Evolutionary sequence covariation analysis to identify functionally coupled residues

• Protein function prediction through automated annotation tools (based on structure and sequence)

These computational predictions should guide experimental design by generating testable hypotheses about BCE_1688's function. The complete 184-amino acid sequence provides sufficient information for these computational approaches, which can be iteratively refined as experimental data becomes available, creating a powerful feedback loop between computational prediction and experimental validation.

How does BCE_1688 compare to similar proteins in other Bacillus species and related bacteria?

BCE_1688 belongs to the UPF0398 protein family, found across various Bacillus species and related genera. Comparative genomic analysis reveals both conserved and divergent features that can provide insights into its potential function. The protein shows significant sequence homology with orthologs in other Bacillus species, particularly B. thuringiensis and B. anthracis, which together with B. cereus form the B. cereus group with highly similar chromosomal backgrounds .

When comparing these proteins:

• The core structural domains are typically conserved, suggesting preservation of fundamental functions

• Species-specific variations often occur in surface-exposed regions, potentially reflecting adaptation to different ecological niches

• Genomic context analysis reveals that BCE_1688 orthologs frequently maintain similar neighboring genes across related species, indicating potential functional relationships or operon structures

This conservation pattern differs markedly from known virulence factors like hemolysin BL (HBL) and non-hemolytic enterotoxin (NHE), which show greater variability across strains and species . The high conservation of BCE_1688 suggests it may serve a fundamental physiological role rather than a specialized virulence function, though this doesn't exclude potential contributions to pathogenicity. Researchers can leverage this comparative information to design experiments that test functional hypotheses derived from better-characterized orthologs in related species.

What experimental design strategies should researchers consider when comparing BCE_1688 with toxic components of Bacillus cereus?

When designing experiments to compare BCE_1688 with established toxic components of B. cereus, researchers should implement a systematic, multi-level comparative analysis framework. Unlike characterized toxins such as hemolysin BL (HBL), which consists of three components (B, L₁, and L₂) and is known to activate the NLRP3 inflammasome , BCE_1688's function remains unclear and requires careful experimental design to evaluate potential toxicity.

A comprehensive experimental strategy should include:

• Parallel purification and characterization of BCE_1688 and known toxins (like HBL components) under identical conditions to enable direct comparisons

• Comparative cytotoxicity assays using multiple cell types (epithelial cells, macrophages, erythrocytes) with concentration gradients to establish dose-response relationships

• Side-by-side evaluation of inflammasome activation, measuring caspase-1 activation, IL-1β/IL-18 secretion, and pyroptosis induction, with NLRP3-deficient cells as controls

• Membrane interaction studies comparing pore-forming capabilities, as HBL is known to form membrane pores leading to potassium efflux and NLRP3 activation

• Neutralization experiments with specific antibodies to distinguish between effects caused by BCE_1688 versus contaminating toxins

Additionally, researchers should generate isogenic bacterial strains with specific deletions (ΔBCE_1688, ΔHBL) and combinations thereof to assess their relative contributions to virulence in cellular and animal models. This comparative approach will help position BCE_1688 within the spectrum of B. cereus virulence factors and determine whether it functions independently or cooperatively with established toxins.