Recombinant Bacillus pseudofirmus UPF0754 protein BpOF4_11355 (BpOF4_11355)

What is the structure and function of BpOF4_11355 protein?

BpOF4_11355 is a full-length (1-379 amino acids) recombinant protein from Bacillus pseudofirmus that belongs to the UPF0754 protein family. While its precise function remains under investigation, structural analysis suggests potential roles in cellular processes typical of this alkaliphilic bacterium . To determine structure-function relationships, researchers should consider:

• X-ray crystallography to resolve three-dimensional structure

• Circular dichroism spectroscopy to analyze secondary structure elements

• Computational modeling using homology-based approaches to predict functional domains

• Functional assays based on hypothesized biochemical activities

The recombinant version available for research includes a histidine tag for purification purposes, which should be considered when interpreting structural data or designing experiments .

What expression systems are recommended for BpOF4_11355 protein?

The commercially available BpOF4_11355 protein is expressed in E. coli expression systems . For researchers developing their own expression protocols, consider:

When using E. coli systems, optimization of induction conditions (temperature, IPTG concentration) and lysis protocols are critical for obtaining soluble protein. Consider testing multiple strains (BL21(DE3), Rosetta, Origami) to identify optimal expression conditions for this particular alkaliphile-derived protein.

How should researchers optimize purification of BpOF4_11355 protein?

Given that BpOF4_11355 contains a histidine tag, immobilized metal affinity chromatography (IMAC) is the primary purification method . A comprehensive purification strategy should include:

• Initial capture using Ni-NTA or Co-based IMAC under native or denaturing conditions

• Secondary purification using ion exchange chromatography based on the protein's theoretical pI

• Final polishing step using size exclusion chromatography

• Quality control assessment using SDS-PAGE and Western blotting

The purification buffer composition should account for the alkaliphilic nature of the source organism. Consider testing buffers with pH ranges of 8.0-10.0 to maintain native conformation. For functional studies, determine whether the His-tag affects activity and consider tag removal using appropriate proteases if necessary.

What are recommended approaches for studying BpOF4_11355 protein interactions?

To investigate protein-protein interactions involving BpOF4_11355, researchers should employ multiple complementary techniques:

When designing these experiments, consider both the His-tag's potential interference with interactions and the native cellular environment of Bacillus pseudofirmus. Control experiments should include testing interactions with known bacterial proteins from similar pathways to validate experimental conditions .

How should researchers design expression constructs for functional studies of BpOF4_11355?

When designing expression constructs for functional characterization, consider these methodological approaches:

• Generate multiple constructs with varying tag positions (N-terminal, C-terminal) to assess tag influence on function

• Include constructs with different tag types (His, GST, MBP) to optimize solubility and activity

• Consider domain-based constructs if bioinformatic analysis predicts multiple functional domains

• Design site-directed mutagenesis constructs targeting predicted catalytic or binding residues

Use codon optimization for the expression system of choice, particularly important when expressing this alkaliphilic bacterial protein in heterologous systems. Include appropriate protease cleavage sites for tag removal if native protein is required for functional assays.

What approaches are recommended for studying BpOF4_11355 in the context of Bacillus pseudofirmus physiology?

To understand the physiological role of BpOF4_11355 in its native context:

• Generate knockout mutants using CRISPR-Cas9 or traditional homologous recombination

• Perform complementation studies with wild-type and mutant variants

• Conduct phenotypic assays under different environmental conditions (pH variations, salt stress)

• Analyze global effects of gene deletion using transcriptomics or proteomics

When designing these experiments, consider the alkaliphilic nature of Bacillus pseudofirmus and ensure that growth conditions reflect its natural environment. Control experiments should include monitoring growth rates, morphology, and stress responses under standard and challenge conditions.

What are the recommended methods for assessing purity and quality of BpOF4_11355 preparations?

Quality control is essential for reliable research results. For BpOF4_11355, consider this analytical workflow:

Ensure that quality control data is thoroughly documented for reproducibility. The molecular weight calculation should account for the full-length protein (379 amino acids) plus the His-tag . Consider establishing release criteria for protein preparations based on these analytical methods.

How can researchers troubleshoot low expression or insolubility issues with BpOF4_11355?

When facing challenges with BpOF4_11355 expression or solubility, implement this systematic troubleshooting approach:

• Expression optimization:

  • Test multiple E. coli strains (BL21, Rosetta, Arctic Express)

  • Vary induction conditions (0.1-1.0 mM IPTG, 16-37°C)

  • Try auto-induction media for gradual protein expression

• Solubility enhancement:

  • Co-express with molecular chaperones (GroEL/ES, DnaK/J)

  • Test fusion partners (MBP, SUMO, TrxA) known to improve solubility

  • Optimize lysis buffer composition (detergents, salt concentration)

• Refolding strategies if inclusion bodies persist:

  • Develop on-column refolding protocols during IMAC purification

  • Test stepwise dialysis with decreasing denaturant concentrations

  • Screen additives (L-arginine, glycerol, sucrose) that promote folding

Monitor results using SDS-PAGE analysis of soluble and insoluble fractions at each optimization step.

What techniques are recommended for assessing functional activity of BpOF4_11355?

Without established functional assays for this specific protein, researchers should develop activity tests based on bioinformatic predictions:

• If sequence analysis suggests enzymatic activity:

  • Design substrate screening assays for hydrolase, transferase, or other predicted activities

  • Monitor potential cofactor requirements (metal ions, nucleotides)

  • Test activity under various pH conditions (pH 7-11) reflecting the alkaliphilic nature

• If structural analysis suggests binding functions:

  • Perform ligand binding assays using thermal shift assays

  • Conduct isothermal titration calorimetry with potential binding partners

  • Use microscale thermophoresis for quantitative binding analysis

• For all functional assessments:

  • Include appropriate positive and negative controls

  • Compare wild-type and mutant protein variants

  • Test activity under conditions mimicking Bacillus pseudofirmus cellular environment

How can researchers investigate the role of BpOF4_11355 in specific cellular pathways?

To elucidate pathway involvement, implement these advanced research strategies:

• Interactome analysis:

  • Perform BioID or APEX proximity labeling with BpOF4_11355 as bait

  • Conduct quantitative proteomics to identify pathway components

  • Validate key interactions using co-immunoprecipitation or FRET

• Functional genomics:

  • Analyze phenotypic consequences of gene deletion under various conditions

  • Perform transcriptomic analysis comparing wild-type and knockout strains

  • Conduct synthetic lethal screens to identify genetic interactions

• Metabolomic analysis:

  • Compare metabolite profiles between wild-type and mutant strains

  • Track metabolic fluxes using stable isotope labeling

  • Correlate metabolic changes with phenotypic observations

These approaches should be conducted under conditions relevant to Bacillus pseudofirmus physiology, particularly considering its alkaliphilic nature.

What approaches are recommended for structural characterization of BpOF4_11355?

For comprehensive structural characterization, consider this hierarchical approach:

Begin with computational approaches to guide experimental design. For experimental methods, ensure protein purity exceeds 95% and assess sample homogeneity using dynamic light scattering before structural studies. Consider the impact of the His-tag on structure and remove it if necessary for high-resolution studies .

How can researchers investigate post-translational modifications of BpOF4_11355?

Although bacterial proteins typically have fewer post-translational modifications (PTMs) than eukaryotic proteins, several approaches can identify potential modifications:

• Mass spectrometry-based analysis:

  • Perform bottom-up proteomics with multiple proteases for comprehensive coverage

  • Use electron transfer dissociation for labile modification detection

  • Implement targeted approaches for specific modifications (phosphorylation, methylation)

• Site-specific analysis:

  • Generate antibodies against predicted modified peptides

  • Perform site-directed mutagenesis of potential modification sites

  • Use chemical labeling strategies for specific modifications

• Functional correlation:

  • Compare activity of protein expressed in different systems (E. coli vs. B. pseudofirmus)

  • Assess activity changes under conditions that might regulate modifications

  • Test modification-mimicking mutations (e.g., phosphomimetics)

When analyzing PTMs, consider the native environment of Bacillus pseudofirmus, particularly its alkaliphilic nature which may influence modification patterns.

How should researchers document experiments with BpOF4_11355 for maximum reproducibility?

To ensure reproducibility in BpOF4_11355 research, implement these comprehensive documentation practices:

• Experimental materials documentation:

  • Record complete source information (strain, vector, expression system)

  • Document exact buffer compositions, including pH and additives

  • Maintain detailed records of all reagents and their lot numbers

• Methodological documentation:

  • Create detailed protocols with timing, temperatures, and equipment settings

  • Record any deviations from standard protocols

  • Document equipment calibration and validation data

• Data collection and analysis:

  • Maintain raw data files in non-proprietary formats where possible

  • Document all data processing steps and parameters

  • Record statistical analysis methods and justification

Consider adopting electronic laboratory notebook systems with version control to ensure complete experiment tracking. This approach facilitates both internal reproducibility and method sharing with the broader research community.

What reporting standards should be followed when publishing research on BpOF4_11355?

When preparing manuscripts on BpOF4_11355 research, adhere to these reporting guidelines:

Include sufficient methodological detail for other researchers to reproduce the work. Use appropriate tables and figures to present data efficiently while maintaining clarity . Ensure proper citation of sources and methods, particularly for adapted protocols. Consider publishing detailed protocols in method-focused journals or repositories to supplement research articles.