Recombinant Bacillus cereus UPF0302 protein BCE33L1402 (BCE33L1402)

What is the UPF0302 protein BCE33L1402 from Bacillus cereus?

BCE33L1402 belongs to the UPF0302 family of uncharacterized proteins found in Bacillus cereus. As a member of the UPF (Uncharacterized Protein Family) category, its precise biological function remains to be fully elucidated. Based on approaches used for similar B. cereus proteins, functional characterization would typically involve sequence alignment, phylogenetic analysis, and comparison with related proteins from other Bacillus species. Similar methodologies for categorizing uncharacterized proteins have been applied to other B. cereus proteins, such as the BC3310 efflux protein, which was classified using bioinformatics tools including MUSCLE alignment and phylogenetic tree construction using MrBayes .

What expression systems are most suitable for recombinant production of BCE33L1402?

For recombinant expression of B. cereus proteins, several systems have proven effective. Based on methodologies applied to similar proteins, E. coli expression systems using vectors such as pTTQ18 (as used for BC3310) can be employed for initial characterization . For native-like expression, homologous expression within Bacillus systems has shown success. For instance, the study on Bacillus cereus demonstrated successful heterologous expression of cellulase under the control of a protease promoter, achieving activity levels of 0.61 u.mL⁻¹ before optimization . For BCE33L1402, a similar approach could be implemented, potentially using the aprE promoter system that has shown success in other B. cereus recombinant protein expressions.

How can I confirm the successful expression of recombinant BCE33L1402?

Verification of successful expression typically involves multiple techniques. For proteins expressed with tags (such as His-tags), Western blotting using anti-His antibodies provides a reliable detection method. Additionally, functional assays specific to the protein family can confirm activity, though these may be limited for uncharacterized proteins. For B. cereus proteins, researchers have employed techniques such as colony PCR to verify gene integration, followed by activity assays when applicable . For BCE33L1402, PCR verification of gene insertion combined with protein purification and SDS-PAGE analysis would provide confirmation of successful expression, similar to the approach used for other B. cereus recombinant proteins.

What strategies can improve the expression yield of BCE33L1402 in Bacillus cereus?

Several advanced strategies have been demonstrated to enhance recombinant protein expression in B. cereus. One particularly effective approach involves the manipulation of transcriptional regulators. Research has shown that deletion of the scoC gene, a major transcriptional regulator in Bacillus species, can significantly increase protein production. In one study, scoC deletion in B. cereus increased protease activity from 230 u.mL⁻¹ to 363.14 u.mL⁻¹ (approximately 58% increase) and simultaneously enhanced cellulase activity by 28% . For BCE33L1402, a similar genetic manipulation approach could be implemented, targeting relevant transcriptional regulators after identifying the regulatory elements controlling BCE33L1402 expression.

What are the most effective methods for functional characterization of an uncharacterized protein like BCE33L1402?

Functional characterization of uncharacterized proteins requires a multi-faceted approach. For BCE33L1402, an effective research strategy would include:

• In silico analysis: Sequence alignment with characterized proteins, domain prediction, and structural modeling. Similar approaches were used for BC3310, which was classified as a member of the major facilitator superfamily through in silico analysis .

• Deletion mutant analysis: Construction of a markerless mutant (as described for bc3310) allows for phenotypic comparison with wild-type strains . For BCE33L1402, this would involve creating a deletion strain following methodologies similar to those described by Simm et al. (2012) and Janes and Stibitz (2006) .

• Overexpression studies: Heterologous expression in a well-characterized host allows for isolation and characterization of the protein's biochemical properties in vitro . For BCE33L1402, this could be achieved using expression vectors like pTTQ18, as utilized for BC3310 .

• Interactome studies: Identifying protein-protein interactions can provide insights into function within cellular pathways.

How can I determine the stability and structural characteristics of BCE33L1402?

Protein stability and structural characterization are critical for understanding function. For BCE33L1402, a systematic approach would include:

• Bioinformatic prediction: Tools like the instability index calculation used for cellulase and protease in B. cereus (which yielded values of 26.16 and 20.18 respectively, indicating stability as they were below the threshold of 40) .

• Thermal stability assays: Differential scanning fluorimetry or thermal shift assays to determine melting temperatures.

• Circular dichroism: To assess secondary structure components.

• Limited proteolysis: To identify stable domains and flexible regions.

• Crystallography or cryo-EM: For detailed structural analysis if preliminary studies indicate significant research potential.

What promoter systems work best for BCE33L1402 expression in Bacillus cereus?

The choice of promoter significantly impacts recombinant protein expression. For B. cereus proteins, several promoter systems have demonstrated efficacy:

The aprE promoter system has shown particular promise in B. cereus, especially when combined with scoC deletion to remove repressive effects on gene expression .

What are the optimal conditions for transforming BCE33L1402 constructs into Bacillus cereus?

Transformation efficiency is crucial for successful recombinant protein studies. For B. cereus, natural transformation has shown surprising efficiency in some strains. The B. cereus strain used in the referenced study demonstrated high potential for DNA uptake and integration into its genome, with approximately 28% efficiency . This property is particularly valuable for genetic manipulation.

For BCE33L1402 transformation, the following protocol could be adapted from successful B. cereus transformations:

• Prepare competent cells in modified Spizizen's solution (glucose 0.5%, tryptone 0.01%, yeast extract 0.1%, MgSO4·7H2O 0.2%, Spizizen's Salts) .

• Incubate at 37°C and 100 × g for 3 hours to induce natural competence.

• Add purified DNA (~1 μg with A260/280 ratio of ~1.79) to competent cells.

• Incubate at 37°C and 180 × g for 2 hours.

• Plate appropriate dilutions on selective media .

What purification strategy is most effective for BCE33L1402?

Purification strategies depend on the protein's properties and expression system. For BCE33L1402, a systematic approach would include:

• If expressed with affinity tags (e.g., His-tag): Immobilized metal affinity chromatography (IMAC) provides an efficient first purification step, similar to the approach used for other B. cereus proteins .

• For secreted BCE33L1402: If expressed under the aprE promoter system, the protein may be secreted into the culture medium, simplifying the initial purification steps as demonstrated for other B. cereus extracellular enzymes .

• Further purification: Size exclusion chromatography and/or ion exchange chromatography to achieve higher purity.

• Quality control: SDS-PAGE, Western blotting, and mass spectrometry to confirm purity and identity.

How can I assess the biochemical properties of purified BCE33L1402?

Biochemical characterization is essential for understanding protein function. For BCE33L1402, a comprehensive analysis would include:

• Activity assays: While specific functions are unknown for UPF0302 proteins, general biochemical tests (ATPase activity, DNA/RNA binding, etc.) can provide initial insights.

• Stability analysis: pH and temperature profiles to determine optimal conditions, similar to the optimization performed for other B. cereus enzymes .

• Structural analysis: Secondary structure analysis via circular dichroism, followed by more detailed structural studies if warranted.

• Interaction studies: Pull-down assays or co-immunoprecipitation to identify potential binding partners.

What approaches can be used to create BCE33L1402 deletion mutants in Bacillus cereus?

Creating deletion mutants is valuable for functional studies. For BCE33L1402, successful approaches include:

• Markerless deletion: Using the method of Janes and Stibitz (2006) as applied for the bc3310 gene . This involves:

  • Amplifying upstream and downstream regions of BCE33L1402 using specific primers

  • Joining these regions through SOEing PCR to create a deletion construct

  • Transforming the construct into B. cereus

  • Selecting for homologous recombination events

• Homologous recombination: B. cereus strains have shown high efficiency (28%) for recombination using PCR products with short homologous arms . This property is particularly useful for genetic manipulation of BCE33L1402.

• CRISPR-Cas9 systems: While not mentioned in the search results, CRISPR-based approaches have been adapted for Bacillus species and could provide an efficient alternative for BCE33L1402 deletion.

How can I construct BCE33L1402 point mutants to study structure-function relationships?

Site-directed mutagenesis of BCE33L1402 would follow standard molecular biology techniques:

• Using plasmid-based systems similar to the pTTQ18 system used for BC3310 .

• Implementing PCR-based mutagenesis protocols to introduce specific mutations.

• Confirming mutations through sequencing before expression and characterization.

• Systematic mutation of conserved residues identified through sequence alignment with related proteins.