Recombinant Lysinibacillus sphaericus UPF0754 membrane protein Bsph_0374 (Bsph_0374)

What is the molecular structure of Bsph_0374?

Bsph_0374 is a UPF0754 membrane protein from Lysinibacillus sphaericus with a full amino acid sequence length of 380 residues (expression region 1-380) . The protein has a UniProt accession number of B1HVI2 and is classified as a membrane protein . Its amino acid sequence contains multiple hydrophobic regions consistent with membrane-spanning domains, including the N-terminal sequence "MDNFIVTLLFMAIIGAAIGGVTNHLAIK" which likely represents a transmembrane segment . For structural analysis, researchers should consider utilizing techniques like circular dichroism spectroscopy or X-ray crystallography to determine secondary and tertiary structural elements.

What is known about the host organism Lysinibacillus sphaericus?

Lysinibacillus sphaericus (formerly classified as Bacillus sphaericus) is a Gram-positive, aerobic, spore-forming bacterium commonly isolated from soil environments . The bacterium is notable for producing mosquitocidal binary toxins (Bin toxins) that are deposited within a balloon-like exosporium during sporulation . L. sphaericus forms spores with distinctive structural properties, including an exosporium that differs from related Bacillus species. Understanding the biological context of L. sphaericus provides important background for researchers investigating membrane proteins like Bsph_0374 from this organism.

How should recombinant Bsph_0374 be stored for optimal stability?

For maintaining optimal stability of recombinant Bsph_0374, the protein should be stored at -20°C in a Tris-based buffer containing 50% glycerol optimized for this specific protein . For extended storage periods, it is recommended to conserve the protein at -20°C or -80°C . Researchers should avoid repeated freeze-thaw cycles as this may compromise protein integrity. Working aliquots can be maintained at 4°C for up to one week to minimize degradation from repeated temperature changes . When designing experiments, consider performing stability tests under various storage conditions if extended work with the protein is planned.

What expression systems are optimal for producing recombinant Bsph_0374?

Recombinant Bsph_0374 has been successfully expressed in E. coli expression systems with His-tag modifications . When designing an expression strategy, researchers should consider:

• Vector selection: Vectors with strong promoters (T7, tac) generally work well for membrane protein expression

• Host strain optimization: BL21(DE3), C41, or C43 strains often perform better for membrane proteins

• Induction conditions: Lower temperatures (16-25°C) and reduced IPTG concentrations may improve folding

• Membrane extraction techniques: Detergent screening to identify optimal solubilization conditions

For membrane proteins like Bsph_0374, expression level optimization requires balancing protein production with proper membrane insertion and folding. A systematic approach comparing different expression conditions would involve the following experimental design:

What purification strategies are effective for Bsph_0374?

Given that Bsph_0374 is available as a His-tagged recombinant protein , immobilized metal affinity chromatography (IMAC) represents the primary purification strategy. A comprehensive purification protocol should include:

• Cell lysis optimization: Mechanical disruption methods (sonication, homogenization) combined with detergent solubilization

• IMAC purification: Using Ni-NTA or Co-NTA resins with imidazole gradient elution

• Secondary purification: Size exclusion chromatography to remove aggregates and achieve higher purity

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

For membrane proteins, detergent selection is critical. Researchers should test multiple detergents (DDM, LDAO, FC-12) for optimal solubilization while maintaining protein structure and function.

How can the membrane topology of Bsph_0374 be experimentally determined?

Determining the membrane topology of Bsph_0374 requires specialized techniques that identify which protein regions are exposed to different cellular compartments. An effective experimental approach would include:

• Cysteine scanning mutagenesis: Introducing cysteine residues at various positions followed by accessibility labeling

• Protease protection assays: Limited proteolysis of membrane-inserted protein to identify exposed regions

• Fluorescence techniques: Fusion of GFP/mCherry at various positions to determine localization, similar to approaches used for other L. sphaericus proteins

• Computational prediction validation: Comparing experimental results with predictions from algorithms like TMHMM or Phobius

For a comprehensive analysis, researchers could adapt the methodological approach used for other membrane proteins from L. sphaericus, as demonstrated in the case of BclS protein, where fusion protein visualization revealed dynamic localization patterns .

How does Bsph_0374 compare to other UPF0754 family proteins?

Conducting a comparative analysis of Bsph_0374 with other UPF0754 family proteins would involve:

• Sequence alignment: Using tools like BLAST, ClustalW, or MUSCLE to identify conserved regions

• Phylogenetic analysis: Constructing trees to understand evolutionary relationships

• Domain structure comparison: Identifying functional motifs or domains shared among family members

• Structural modeling: Generating homology models based on related proteins with known structures

Researchers should note that UPF (Uncharacterized Protein Family) designations indicate limited functional characterization. Therefore, comparative genomic approaches and structure-function studies are particularly valuable for these protein families.

What potential functional roles might Bsph_0374 play in L. sphaericus?

While specific functional data for Bsph_0374 is limited in the available research, the following hypotheses can be generated based on its classification as a membrane protein:

• Cell envelope integrity: Membrane proteins often contribute to maintaining cellular structure

• Transport functions: Potential involvement in nutrient uptake or waste export

• Signal transduction: Possible role in sensing environmental conditions

• Spore formation: Given L. sphaericus' spore-forming nature, potential involvement in sporulation processes similar to other membrane proteins in this organism

To investigate these potential functions, researchers could design knockout experiments using techniques similar to those described for other L. sphaericus proteins, such as the allele replacement methodology used for BclS studies . Phenotypic analyses examining growth rates, membrane integrity, stress responses, and sporulation efficiency would provide insights into the protein's functional role.

How might Bsph_0374 interact with other proteins in L. sphaericus?

To identify protein-protein interactions involving Bsph_0374, researchers should consider:

• Co-immunoprecipitation assays: Using anti-His antibodies to pull down Bsph_0374 complexes

• Bacterial two-hybrid systems: Testing specific interaction partners

• Cross-linking studies: Chemical cross-linking followed by mass spectrometry

• Proximity labeling: BioID or APEX2 fusion proteins to identify neighboring proteins

When analyzing potential interacting partners, researchers should systematically assess both direct physical interactions and functional associations. Interaction data could be organized as follows:

What membrane technology approaches are relevant for studying Bsph_0374?

Membrane proteins like Bsph_0374 can be studied using various membrane technology approaches. These methodologies can be classified based on their molecular weight cut-off (MWCO) and membrane characteristics:

These membrane technologies can be applied to:

• Concentrate dilute protein solutions

• Exchange buffers for functional assays

• Remove contaminants based on size differences

• Prepare protein-containing liposomes for functional studies

How can researchers design experiments to test temperature sensitivity of Bsph_0374?

To investigate the temperature sensitivity of Bsph_0374, researchers can adapt protocols similar to those used for studying L. sphaericus spore heat resistance . A comprehensive experimental design would include:

• Sample preparation: Purified Bsph_0374 in appropriate buffer systems

• Temperature range testing: Systematic exposure to temperatures from 4°C to 80°C

• Duration variables: Short (minutes) and extended (hours) exposure times

• Analytical methods: Circular dichroism to monitor secondary structure changes, activity assays to assess functional preservation

Experimental data should be organized in clear tables following scientific data presentation guidelines:

These data points would provide valuable insights into protein stability thresholds for experimental design .

What approaches can be used to investigate the role of Bsph_0374 in L. sphaericus physiology?

To investigate the physiological role of Bsph_0374, researchers could employ gene deletion strategies similar to those used for other L. sphaericus proteins . A comprehensive approach would include:

• Gene knockout construction: Using allele replacement methods with kanamycin resistance markers

• Complementation studies: Reintroducing the native gene to confirm phenotype restoration

• Phenotypic characterization:

  • Growth curves under various conditions

  • Membrane integrity assays

  • Stress resistance testing

  • Sporulation efficiency assessment

For spore-related phenotypes, researchers could adapt protocols used for BclS studies, including microscopy techniques, heat resistance measurements, and germination rate determination . The methodological approach should include appropriate controls and multiple biological replicates to ensure reproducibility.

How should researchers interpret contradictory results in Bsph_0374 functional studies?

When faced with contradictory results in Bsph_0374 studies, researchers should:

• Examine methodological differences: Variations in protein preparation, buffer conditions, or analytical techniques

• Consider post-translational modifications: Differences in expression systems may affect protein modifications

• Evaluate protein conformational states: Membrane proteins often exist in multiple conformations affecting function

• Assess experimental conditions: pH, temperature, salt concentration, and detergent effects

A systematic approach to resolving contradictions involves comparative experiments under standardized conditions, using multiple analytical techniques to corroborate findings. Researchers should maintain detailed records of all experimental variables to facilitate troubleshooting.

What are common challenges in membrane protein research applicable to Bsph_0374 studies?

Membrane protein research, including work with Bsph_0374, presents several challenges:

• Expression obstacles:

  • Low expression yields due to cytotoxicity

  • Inclusion body formation requiring refolding

  • Membrane insertion efficiency limitations

• Purification difficulties:

  • Detergent selection affecting stability and function

  • Protein aggregation during concentration

  • Lipid requirements for structural integrity

• Structural analysis limitations:

  • Challenges in obtaining diffraction-quality crystals

  • Detergent micelle interference in structural studies

  • Dynamic conformational changes difficult to capture

For each challenge, researchers should implement mitigation strategies such as screening multiple expression conditions, testing various detergents, and employing complementary structural analysis techniques. Documentation of successful approaches in laboratory notebooks will benefit future studies.

What are promising future research directions for Bsph_0374?

Given the current state of knowledge about Bsph_0374, several promising research directions emerge:

• Structural characterization: Determining high-resolution structures using cryo-EM or X-ray crystallography

• Functional annotation: Identifying biochemical activities and physiological roles

• Interactome mapping: Characterizing the protein interaction network

• Evolutionary analysis: Understanding conservation patterns across bacterial species

• Applied research: Exploring potential biotechnological applications based on functional properties

Researchers entering this field should consider interdisciplinary approaches combining genomics, proteomics, structural biology, and microbial physiology to develop a comprehensive understanding of this uncharacterized membrane protein.

How can researchers contribute to the broader understanding of UPF0754 family proteins?

To advance knowledge of the UPF0754 protein family:

• Develop standardized protocols for expression and purification

• Create a database of characterized family members and their properties

• Establish collaborative networks to share resources and expertise

• Apply systems biology approaches to identify functional networks

• Utilize comparative genomics to predict functional roles