Recombinant Bacillus cereus UPF0344 protein BC_1150 (BC_1150)

What is the basic structure and characteristics of the BC_1150 protein?

The BC_1150 protein is a full-length protein (1-121 amino acids) from Bacillus cereus with the UniProt ID Q81GP1. Its complete amino acid sequence is MVHMHITAWALGLILFFVAYSLYSAGRKGKGVHMGLRLMYIIIIVTGFMLYMSIVKTATGSMHMWYGLKMLAGILVIGGMEMVLVKMSKNKPTGAVWGLFIVALVAVFYLGLKLPIGWKVF . Based on its sequence characteristics, it appears to be a membrane protein with multiple hydrophobic regions, suggesting potential transmembrane domains. The protein belongs to the UPF0344 family, which consists of proteins with unknown function that share sequence similarity.

How should BC_1150 protein be stored and reconstituted for experimental use?

For optimal stability, store the lyophilized BC_1150 protein powder at -20°C/-80°C upon receipt . It's recommended to briefly centrifuge the vial before opening to ensure the content is at the bottom. Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL . For long-term storage, add glycerol to a final concentration of 5-50% (typically 50% is recommended) and aliquot before storing at -20°C/-80°C .

For working solutions, aliquots can be stored at 4°C for up to one week, but repeated freeze-thaw cycles should be avoided as they can compromise protein integrity . The protein is typically provided in a Tris/PBS-based buffer with 6% Trehalose, pH 8.0 , or a Tris-based buffer with 50% glycerol optimized for the protein .

What expression systems are suitable for recombinant BC_1150 production?

Based on the available information, E. coli has been successfully used as an expression system for recombinant BC_1150 production . The full-length protein (amino acids 1-121) can be expressed with an N-terminal His tag to facilitate purification. While E. coli is the most documented system, researchers might consider exploring other expression systems used for Bacillus proteins, such as Bacillus subtilis, which has been successfully used for other Bacillus cereus proteins .

When designing expression strategies, it's important to consider the specific requirements of membrane proteins if structural studies are planned, as BC_1150 appears to have transmembrane domains based on its sequence.

What purification methods are recommended for recombinant BC_1150?

The His-tagged version of BC_1150 can be purified using standard immobilized metal affinity chromatography (IMAC) techniques . Based on protocols used for other recombinant Bacillus proteins, a multi-step purification process might include:

• Initial capture using nickel or cobalt-based IMAC

• Intermediate purification using ion exchange chromatography

• Polishing step using size exclusion chromatography

The specific buffer compositions should be optimized based on protein stability and downstream applications. For example, a study on phospholipase C from Bacillus cereus employed a three-step purification process that yielded highly pure protein with specific activity of 13,190 U mg⁻¹ . Similar principles could be applied to BC_1150 purification, with appropriate modifications based on the protein's properties.

What approaches can be used to determine the function of the UPF0344 protein BC_1150?

As a protein of unknown function (UPF), determining BC_1150's biological role requires multiple complementary approaches:

• Bioinformatic analysis: Perform sequence-based predictions of structure and function using tools like Phyre2, I-TASSER, or AlphaFold2. Identify conserved domains and sequence similarities with proteins of known function.

• Structural studies: Determine the 3D structure using X-ray crystallography, cryo-EM, or NMR spectroscopy to gain insights into potential function based on structural homology.

• Protein-protein interaction studies: Identify binding partners using techniques such as co-immunoprecipitation, yeast two-hybrid screening, or proximity labeling (BioID, APEX).

• Gene knockout/knockdown experiments: Create BC_1150 deletion mutants in Bacillus cereus and characterize phenotypic changes. Similar approaches to those used in the scoC deletion experiments in Bacillus cereus could be applied .

• Heterologous expression studies: Express BC_1150 in different hosts and assess phenotypic changes or functional activities, similar to the methodology used for cellulase expression in B. cereus .

• Transcriptomic and proteomic analyses: Investigate expression patterns of BC_1150 under different conditions to identify potential functional associations.

How can protein-membrane interactions of BC_1150 be characterized?

Given the hydrophobic regions in BC_1150's sequence suggesting potential membrane association, several techniques can be employed to characterize these interactions:

• Detergent screening: Test various detergents for their ability to solubilize BC_1150 from membranes, which can provide insights into the strength and nature of membrane interactions.

• Liposome binding assays: Reconstitute BC_1150 with different lipid compositions to determine specific lipid preferences and binding kinetics.

• Fluorescence techniques: Use techniques such as Förster resonance energy transfer (FRET) or fluorescence correlation spectroscopy (FCS) with fluorescently labeled BC_1150 to study its membrane dynamics.

• Membrane topology mapping: Employ cysteine scanning mutagenesis combined with accessibility assays, or protease protection assays, to determine the orientation of BC_1150 in membranes.

• Molecular dynamics simulations: Conduct in silico analyses of BC_1150's interactions with model membranes to predict structural arrangements and energetically favorable conformations.

What strategies can optimize expression yield and solubility of recombinant BC_1150?

For optimizing expression and solubility of BC_1150, consider the following strategies:

• Expression vector optimization: Test different promoters, signal sequences, and fusion partners. For Bacillus proteins, an acetoin-controlled expression system has shown success for high-level expression .

• Host strain selection: Compare expression levels in different E. coli strains (BL21(DE3), C41(DE3), C43(DE3), Rosetta) or consider Bacillus subtilis WB800 as used for other Bacillus cereus proteins .

• Culture conditions optimization: Systematically vary temperature, induction timing, inducer concentration, and media composition. For example, when working with similar proteins:

*Optimization required for specific research conditions

• Solubility enhancement: For improved solubility, consider co-expression with molecular chaperones, addition of solubility-enhancing additives (glycerol, arginine), or mild detergents for membrane proteins.

• Refolding strategies: If BC_1150 forms inclusion bodies, develop refolding protocols using gradual dialysis or on-column refolding techniques.

How can post-translational modifications of BC_1150 be identified and characterized?

While bacterial proteins typically have fewer post-translational modifications (PTMs) than eukaryotic proteins, they can still undergo modifications that affect function. To investigate potential PTMs in BC_1150:

• Mass spectrometry-based approaches: Use high-resolution MS combined with enrichment techniques to identify PTMs. Techniques include:

  • Bottom-up proteomics with enzymatic digestion

  • Top-down proteomics analyzing intact proteins

  • Targeted approaches focusing on specific modifications

• Site-directed mutagenesis: Mutate potential modification sites and assess functional consequences to determine the significance of specific PTMs.

• Western blotting: Use modification-specific antibodies (e.g., anti-phospho, anti-acetyl) to detect and quantify PTMs.

• Chemical labeling strategies: Employ techniques like isotope-coded affinity tags (ICAT) or isobaric tags for relative and absolute quantitation (iTRAQ) for quantitative PTM analysis.

• Bioinformatic prediction: Use algorithms to predict potential PTM sites based on sequence motifs and structural features.

What is the recommended protocol for heterologous expression of BC_1150 in E. coli?

Based on successful expression strategies for recombinant Bacillus proteins, the following protocol is recommended:

• Cloning:

  • Design primers to amplify the BC_1150 gene with appropriate restriction sites

  • Clone into a pET-based vector with an N-terminal His tag

  • Verify the construct by sequencing

• Transformation and Expression:

  • Transform the construct into E. coli BL21(DE3) cells

  • Grow transformed cells in LB medium at 37°C until OD600 reaches 0.6-0.8

  • Induce protein expression with 0.5 mM IPTG

  • Continue growth at 25-30°C for 4-6 hours or 18°C overnight

  • Harvest cells by centrifugation at 5000 × g for 15 minutes

• Cell Lysis and Protein Extraction:

  • Resuspend cell pellet in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM PMSF)

  • Lyse cells by sonication or French press

  • Clarify lysate by centrifugation at 15,000 × g for 30 minutes

  • If BC_1150 is in inclusion bodies, additional solubilization steps with detergents or denaturants may be required

• Purification:

  • Load clarified lysate onto a Ni-NTA column equilibrated with binding buffer

  • Wash with buffer containing 20-30 mM imidazole

  • Elute BC_1150 with buffer containing 250-300 mM imidazole

  • Perform buffer exchange to remove imidazole using dialysis or gel filtration

• Quality Control:

  • Verify purity by SDS-PAGE (should be >90%)

  • Confirm identity by Western blotting or mass spectrometry

  • Assess protein folding by circular dichroism or fluorescence spectroscopy

How can researchers troubleshoot low expression or insolubility issues with BC_1150?

When encountering expression or solubility challenges with BC_1150, consider this systematic troubleshooting approach:

• Low Expression Issues:

  • Verify construct sequence for mutations or frame shifts

  • Test different E. coli strains (BL21(DE3), C41(DE3), Rosetta)

  • Optimize codon usage for E. coli

  • Try different induction conditions (temperature, IPTG concentration, induction time)

  • Consider using an auto-induction medium

  • Test alternative expression vectors with different promoters

• Insolubility Issues:

  • Reduce expression temperature to 18-20°C

  • Decrease IPTG concentration to 0.1-0.2 mM

  • Add solubility enhancers to the growth medium (5-10% glycerol, 0.1-0.5% glucose)

  • Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

  • Try fusion tags known to enhance solubility (MBP, SUMO, Thioredoxin)

  • For membrane proteins like BC_1150, add mild detergents (0.1-1% CHAPS, DDM, or Triton X-100)

• Inclusion Body Recovery:

  • If BC_1150 forms inclusion bodies despite optimization, develop a refolding protocol:

    • Solubilize inclusion bodies with 6-8 M urea or 6 M guanidine hydrochloride

    • Perform refolding by gradual dialysis or dilution

    • Use additives that promote folding (L-arginine, glycerol, non-detergent sulfobetaines)

• Alternative Expression Systems:

  • Consider using B. subtilis as an expression host, which has been successful for other B. cereus proteins

  • Design an expression cassette with the protein under control of a native Bacillus promoter, similar to the approach used for cellulase expression

What analytical methods are suitable for studying BC_1150 structure and interactions?

To investigate the structure and interactions of BC_1150, several analytical approaches are recommended:

• Structural Analysis:

  • Circular Dichroism (CD) spectroscopy to determine secondary structure content

  • Nuclear Magnetic Resonance (NMR) for solution structure determination of small domains

  • X-ray crystallography for high-resolution structure (may require removing flexible regions or using crystallization chaperones)

  • Cryo-electron microscopy for membrane-embedded structure

• Interaction Analysis:

  • Surface Plasmon Resonance (SPR) to measure binding kinetics with potential partners

  • Isothermal Titration Calorimetry (ITC) for thermodynamic parameters of interactions

  • Bio-Layer Interferometry (BLI) for real-time binding analysis

  • Pull-down assays using the His-tag for identifying interaction partners from cell lysates

  • Crosslinking Mass Spectrometry (XL-MS) to map interaction interfaces

• Stability and Conformational Analysis:

  • Differential Scanning Fluorimetry (DSF) to assess thermal stability

  • Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS) for oligomeric state determination

  • Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) for conformational dynamics

  • Limited proteolysis to identify stable domains and flexible regions