Recombinant Bacillus anthracis UPF0344 protein BAMEG_3427 (BAMEG_3427)

What is the molecular structure of Recombinant Bacillus anthracis UPF0344 protein BAMEG_3427?

Recombinant Bacillus anthracis UPF0344 protein BAMEG_3427 is a 121-amino acid protein from Bacillus anthracis strain CDC 684/NRRL 3495, identified by UniProt ID C3LBW9. The complete amino acid sequence is: MVHMHITAWALGLILFFVAYSLYSAGRKGKGVHMGLRLMYIIIIVTGFMLYMGIMKTATS NMHMWYGLKMIAGILVIGGMEMVLVKMSKNKATGAVWGLFIVALVAVFYLGLKLPIGWQV F . The protein contains hydrophobic regions suggesting potential membrane association, which is characteristic of many bacterial proteins involved in cellular processes. Structural analysis indicates possible transmembrane domains that may be crucial for its functional properties within the bacterial cell membrane. Preliminary studies suggest a predominantly alpha-helical secondary structure, though comprehensive crystallographic data remains limited for this specific protein.

What is currently known about the function of BAMEG_3427 in Bacillus anthracis?

The specific function of BAMEG_3427 in Bacillus anthracis remains largely uncharacterized compared to other B. anthracis proteins like protective antigen (PA), which has an established role in pathogenicity as a toxin-translocating component . Preliminary analysis of the protein sequence suggests BAMEG_3427 may be membrane-associated, potentially involved in cellular processes related to membrane integrity or transport. Unlike the well-studied exotoxins and capsule components that contribute to B. anthracis virulence , there is currently limited evidence directly linking BAMEG_3427 to pathogenicity mechanisms. The protein's conservation across Bacillus species suggests it may serve a fundamental role in bacterial physiology rather than being specifically involved in virulence. Future functional studies utilizing gene knockout approaches and protein interaction analyses will be critical for elucidating its precise biological role.

What are the optimal expression systems for producing recombinant BAMEG_3427?

For the expression of recombinant BAMEG_3427, Escherichia coli has been demonstrated as an effective heterologous expression system . When designing expression constructs, researchers should consider incorporating an N-terminal His-tag to facilitate subsequent purification steps . The optimal expression vector selection depends on specific research requirements, but arabinose-inducible systems similar to pBAD24 have shown efficacy for expressing Bacillus-derived proteins . Temperature optimization is critical; expression at lower temperatures (16-25°C) often yields higher amounts of soluble protein compared to standard 37°C induction protocols. For membrane-associated proteins like BAMEG_3427, codon optimization for E. coli and the addition of solubility-enhancing fusion partners (such as MBP or SUMO) may increase soluble protein yields. Additionally, considering specialized E. coli strains designed for membrane protein expression (like C41/C43) may improve results for proteins with transmembrane domains.

What purification strategies yield the highest purity for recombinant BAMEG_3427?

Purification of His-tagged recombinant BAMEG_3427 can be achieved using immobilized metal affinity chromatography (IMAC) as the initial capture step . A multi-step purification protocol typically begins with cell lysis in Tris-based buffer (pH 8.0), followed by clarification of the lysate through centrifugation and filtration. For His-tagged constructs, Ni-NTA or TALON resin chromatography with imidazole gradient elution effectively separates the target protein . Secondary purification may include ion exchange chromatography, particularly with HiTrap Q Sepharose XL for further purification . Size exclusion chromatography serves as a polishing step to achieve >90% purity as verified by SDS-PAGE . For membrane-associated proteins like BAMEG_3427, inclusion of mild detergents (0.1% DDM or LDAO) in the purification buffers may improve yields and maintain protein stability. Final purified protein can be stored in Tris/PBS-based buffer containing 6% trehalose at pH 8.0 , with aliquoting recommended to avoid repeated freeze-thaw cycles.

How should researchers optimize storage conditions for maintaining BAMEG_3427 stability?

For optimal stability of purified recombinant BAMEG_3427, storage at -20°C/-80°C is recommended, with -80°C preferred for long-term storage exceeding one month . The protein should be stored in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0, which helps maintain protein structural integrity during freeze-thaw cycles . When preparing for storage, it is critical to aliquot the purified protein to avoid repeated freeze-thaw cycles, as these significantly reduce protein activity and structural integrity . For working stocks, aliquots can be maintained at 4°C for up to one week without significant loss of integrity . When reconstituting lyophilized protein, researchers should use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL . Addition of glycerol to a final concentration of 5-50% is recommended for cryoprotection, with 50% being the standard for optimal preservation . Prior to opening storage vials, brief centrifugation is advised to ensure contents settle at the bottom of the container.

What methodologies are most effective for studying BAMEG_3427 interaction with cellular components?

For investigating BAMEG_3427 interactions with cellular components, several complementary approaches should be employed. Flow cytometry-based binding assays similar to those used for other B. anthracis proteins can assess potential interactions with mammalian cell surface receptors . This technique involves incubating target cells (such as RAW 264.7 macrophages) with the recombinant protein, followed by detection using specific antibodies and fluorescent secondary antibodies . Pull-down assays utilizing the His-tag of recombinant BAMEG_3427 can identify potential binding partners from cell lysates. Biolayer interferometry or surface plasmon resonance provides quantitative binding kinetics measurements. For membrane association studies, liposome binding assays and membrane fractionation techniques are particularly valuable given BAMEG_3427's predicted membrane localization. Fluorescently labeled BAMEG_3427 can be used for microscopy-based localization studies within bacterial or host cells. Cross-linking coupled with mass spectrometry (XL-MS) offers a powerful approach for mapping the protein interaction network of BAMEG_3427 in its native cellular environment.

How can researchers assess the functional activity of recombinant BAMEG_3427?

Assessing the functional activity of recombinant BAMEG_3427 presents challenges due to its uncharacterized nature. A comprehensive approach begins with structural integrity verification using circular dichroism spectroscopy to confirm proper protein folding. Thermal shift assays can determine protein stability under various conditions. Given its potential membrane association, researchers should assess membrane integration using liposome reconstitution assays or nanodiscs. Functional activity may be inferred through comparative assays with other Bacillus species' UPF0344 proteins that have better-characterized functions. ATP release assays, which have proven effective for measuring the lytic activity of other B. anthracis proteins , could be adapted if BAMEG_3427 is suspected to have membrane-perturbing activities. Complementation studies in knockout bacterial strains provide a powerful approach to validate function, where the ability of recombinant BAMEG_3427 to restore wild-type phenotypes in UPF0344-deficient bacteria is assessed. Additionally, electrophysiology techniques may be appropriate if ion channel or transport activities are suspected.

What analytical techniques should be employed to confirm the purity and structural integrity of expressed BAMEG_3427?

A multi-analytical approach should be employed to comprehensively assess the purity and structural integrity of expressed BAMEG_3427. SDS-PAGE remains the primary method for purity assessment, with >90% purity considered acceptable for most research applications . Western blotting using anti-His antibodies confirms identity and integrity of His-tagged constructs. For higher resolution analysis, capillary electrophoresis or analytical size exclusion chromatography can detect minor contaminants or aggregates. Mass spectrometry, particularly electrospray ionization mass spectrometry (ESI-MS), provides precise molecular weight confirmation and can identify post-translational modifications or truncations. Circular dichroism spectroscopy assesses secondary structural elements, confirming proper protein folding. Dynamic light scattering evaluates size distribution and detects potential aggregation. Thermofluor assays can assess thermal stability, which often correlates with proper folding. For membrane-associated proteins like BAMEG_3427, detergent screening using differential scanning fluorimetry helps identify optimal conditions for maintaining native-like structure. Finally, limited proteolysis coupled with mass spectrometry can provide insights into domain organization and accessibility.

How does BAMEG_3427 compare functionally with similar proteins from virulent and non-virulent Bacillus strains?

Comparative functional analysis of BAMEG_3427 with homologs from virulent and non-virulent Bacillus strains represents an important research direction. Unlike well-characterized virulence factors such as protective antigen (PA) that exhibit strain-specific functional variations , the conservation pattern of UPF0344 proteins across the Bacillus genus suggests possible fundamental cellular roles rather than direct virulence functions. Researchers should implement comprehensive phylogenetic analysis coupled with protein structure prediction to identify conserved domains and strain-specific variations. Heterologous expression of UPF0344 homologs from virulent B. anthracis, closely related B. cereus (which includes the representative strain RSVF1 used in comparative studies ), and non-pathogenic Bacillus species enables systematic functional comparison. Cross-complementation experiments in knockout strains can determine functional equivalence across species. Binding studies to cellular components using techniques similar to those employed for PA domains may reveal differential interaction patterns. Comparative transcriptomics analysis under various stress conditions can also highlight potential functional divergence by identifying differential expression patterns of UPF0344 proteins across Bacillus species.

What role might BAMEG_3427 play in Bacillus anthracis pathogenicity compared to established virulence factors?

While established B. anthracis virulence factors like protective antigen (PA) have well-documented roles in pathogenicity as components of anthrax toxin , the potential contribution of BAMEG_3427 to virulence remains largely unexplored. To investigate this question, researchers should compare the expression profiles of BAMEG_3427 and known virulence factors during infection cycles using RNA-seq or quantitative proteomics. Gene knockout studies using CRISPR-Cas9 or homologous recombination techniques can assess the impact of BAMEG_3427 deletion on virulence in appropriate model systems. Researchers should examine potential interactions between BAMEG_3427 and established virulence factors through co-immunoprecipitation or proximity labeling approaches. Cell-binding assays similar to those used for PA domains can determine if BAMEG_3427 interacts with host cells during infection. Mouse infection models comparable to those used for testing PlyG efficacy could evaluate the impact of BAMEG_3427 antibodies or inhibitors on infection progression. Comparative genomics across Bacillus species with varying pathogenicity can reveal evolutionary patterns that might indicate virulence associations. Additionally, structural studies may identify potential binding sites for host factors that could suggest pathogenicity mechanisms.

How can BAMEG_3427 be utilized in developing novel detection systems for Bacillus anthracis?

Development of novel B. anthracis detection systems utilizing BAMEG_3427 represents a promising research direction, building on established approaches used with other bacterial proteins. Researchers can generate highly specific monoclonal antibodies against BAMEG_3427 for use in ELISA-based detection systems similar to those available for other B. anthracis proteins . Species-specific epitopes identified through comparative sequence analysis with UPF0344 proteins from related Bacillus species can enhance detection specificity. Aptamer-based detection systems using SELEX (Systematic Evolution of Ligands by Exponential Enrichment) against purified BAMEG_3427 offer alternatives to antibody-based methods. For rapid field detection, lateral flow immunoassays incorporating anti-BAMEG_3427 antibodies could be developed. Researchers might adapt ATP release assays, which have demonstrated effectiveness for rapid lytic detection of B. anthracis , to create systems triggered by BAMEG_3427 recognition. CRISPR-Cas biosensors targeting BAMEG_3427 genetic sequences offer another innovative approach. To enhance sensitivity, signal amplification techniques such as immuno-PCR or proximity ligation assays can be incorporated. For comprehensive detection systems, researchers should consider combining BAMEG_3427 detection with established markers like protective antigen to create multiplexed assays with improved reliability.

What control experiments are essential when working with recombinant BAMEG_3427?

When designing experiments with recombinant BAMEG_3427, comprehensive controls are essential for result validation. Expression controls should include parallel purification of an unrelated His-tagged protein to distinguish tag-specific from protein-specific effects. Researchers should implement empty vector controls when evaluating expression systems to account for host response to the expression process itself. For functional studies, heat-denatured BAMEG_3427 serves as a negative control to confirm that observed effects require properly folded protein. When conducting binding assays, researchers should include controls similar to those used in PA binding studies, such as secondary antibody-only controls to assess non-specific binding and irrelevant proteins (like Lethal Factor used as a negative control in cell-binding studies ) to confirm binding specificity. For cell-based assays, dose-response experiments with varying protein concentrations establish the relationship between protein amount and observed effects. Time-course studies are necessary to distinguish immediate from delayed effects. When evaluating membrane interactions, liposomes of varying compositions should be tested to determine lipid specificity. Researchers must also include buffer-only controls to account for potential buffer component effects on experimental systems.

How should researchers approach statistical analysis of data obtained from BAMEG_3427 experiments?

Statistical analysis of BAMEG_3427 experimental data requires rigorous approaches to ensure reliability and reproducibility. Before analysis, researchers should establish appropriate sample sizes through power analysis based on expected effect sizes and variability. For basic biochemical assays, a minimum of three independent biological replicates with technical triplicates is recommended. Data normality should be assessed using Shapiro-Wilk or Kolmogorov-Smirnov tests to determine appropriate parametric or non-parametric statistical methods. For comparative binding studies similar to those conducted with PA domains , flow cytometry data should be analyzed using appropriate statistical tests for histogram comparison, with clear reporting of cell counts and fluorescence intensity distributions. When analyzing dose-response relationships, researchers should employ regression analysis to determine EC50/IC50 values with 95% confidence intervals. For complex datasets involving multiple conditions or time points, researchers should consider mixed-effects models to account for repeated measures and potential random effects. Appropriate multiple testing corrections (Bonferroni, Benjamini-Hochberg) should be applied when conducting multiple comparisons. Finally, researchers should report effect sizes alongside p-values to provide context for the biological significance of observed differences.

What are the key considerations for developing antibodies against BAMEG_3427?

Developing specific antibodies against BAMEG_3427 requires careful planning to ensure specificity and utility. Epitope prediction analysis should be conducted to identify immunogenic regions unique to BAMEG_3427, avoiding highly conserved regions that might cross-react with UPF0344 proteins from related Bacillus species. For polyclonal antibody development, researchers should immunize animals with full-length purified recombinant BAMEG_3427 using adjuvant formulations similar to those employed for PA immunization studies (Addavax, Alhydrogel, or Montanide ISA 720) . Multiple immunization boosters are typically required, with serum antibody titers monitored via ELISA. For monoclonal antibody development, after initial immunization, B-cells should be isolated from spleen and fused with myeloma cells to generate hybridomas, followed by screening for specificity using both BAMEG_3427 and related UPF0344 proteins from other Bacillus species. Antibody characterization should include Western blot, immunoprecipitation, flow cytometry, and immunofluorescence applications to ensure versatility. Cross-reactivity testing against proteins from related Bacillus species is essential to confirm specificity. For applications requiring higher specificity, epitope-specific antibodies can be developed using synthetic peptides corresponding to unique regions of BAMEG_3427.

How does the expression and purification of BAMEG_3427 differ from other Bacillus anthracis proteins?

The expression and purification of BAMEG_3427 presents distinct challenges compared to other well-studied B. anthracis proteins. Unlike protective antigen (PA), which has been reported to form inclusion bodies when expressed in E. coli , BAMEG_3427 can be expressed as a soluble protein with appropriate construct design . This contrasts with PA-FL (full-length protective antigen), which researchers have found challenging to express in soluble form, while other PA constructs like PA63, PA-D1-4, and PA-D4 have been successfully expressed solubly . For BAMEG_3427, E. coli expression systems with His-tagging have proven effective , while specialized expression systems might be required for other B. anthracis proteins. Purification strategies for BAMEG_3427 typically involve standard His-tag affinity chromatography followed by size exclusion methods , whereas more complex multi-step purification procedures incorporating ion exchange chromatography like HiTrap Q Sepharose XL have been necessary for other B. anthracis proteins . Additionally, BAMEG_3427's predicted membrane association may necessitate the inclusion of detergents during purification, a consideration not required for soluble B. anthracis proteins like the PA domains used in vaccine studies .

What experimental approaches can determine if BAMEG_3427 interacts with known virulence factors?

To investigate potential interactions between BAMEG_3427 and established B. anthracis virulence factors, researchers should implement a multi-faceted experimental approach. Co-immunoprecipitation studies using antibodies against BAMEG_3427 can identify associated proteins from B. anthracis lysates, with subsequent mass spectrometry to identify potential virulence factor interactions. Yeast two-hybrid screening or bacterial two-hybrid systems can assess direct protein-protein interactions between BAMEG_3427 and virulence factors like protective antigen (PA) or lethal factor (LF). Biolayer interferometry or surface plasmon resonance using purified recombinant proteins provides quantitative binding kinetics. Researchers can employ functional assays similar to those used for PA domains to determine if BAMEG_3427 affects the activity of virulence factors. Fluorescence resonance energy transfer (FRET) using fluorescently labeled proteins can detect interactions in real-time in cellular contexts. Protein crosslinking followed by mass spectrometry (XL-MS) can map interaction interfaces at the amino acid level. For in vivo relevance, co-localization studies using fluorescently tagged proteins can visualize potential interactions within bacterial cells. Genetic approaches, including suppressor screens and synthetic lethality assays, may reveal functional relationships between BAMEG_3427 and virulence factor genes.

How can structural comparison between BAMEG_3427 and other Bacillus proteins inform functional predictions?

Structural comparison between BAMEG_3427 and other Bacillus proteins provides valuable insights for functional prediction. Researchers should begin with in silico structural modeling using homology-based approaches and advanced prediction tools like AlphaFold to generate three-dimensional models of BAMEG_3427. These models can be compared with experimentally determined structures of other Bacillus proteins in the Protein Data Bank. Particular attention should be paid to comparison with functional domains of well-characterized proteins like protective antigen (PA), which has distinct domains (PA-D1, PA-D4) with known functions in receptor binding and toxin translocation . Conserved structural motifs identified through fold recognition algorithms may suggest shared functional properties. Analysis of surface electrostatic potential and hydrophobicity patterns can identify potential binding interfaces or membrane interaction regions. Molecular docking simulations can test hypothesized interactions with potential binding partners or substrates. For experimental validation of structural predictions, researchers should consider limited proteolysis experiments similar to those used to characterize PA domain organization , which can identify stable domains and flexible regions. X-ray crystallography or cryo-electron microscopy of purified BAMEG_3427 would provide definitive structural information, though membrane-associated proteins often present crystallization challenges.