Recombinant Bacillus subtilis Uncharacterized protein ydaN (ydaN)

What is Bacillus subtilis uncharacterized protein ydaN?

Bacillus subtilis ydaN is a protein of interest encoded by the ydaN gene in B. subtilis. It is classified as "uncharacterized" because its complete biological function has not been fully elucidated. The full-length mature protein spans amino acids 24-703 and has a UniProt identifier of O31488. For research purposes, recombinant versions of this protein are typically produced with tags such as His-tags to facilitate purification and detection . Unlike some other characterized B. subtilis proteins such as EndoA (encoded by ydcE gene), which functions as an endoribonuclease in toxin-antitoxin systems , the precise cellular role of ydaN remains under investigation.

How does ydaN protein compare to other characterized B. subtilis proteins?

While ydaN remains uncharacterized, comparing it to well-studied B. subtilis proteins can provide research context. For example, B. subtilis contains characterized proteins like EndoA (encoded by ydcE), which functions as an endoribonuclease within a toxin-antitoxin system . Unlike EndoA, which has a defined enzymatic activity and a partner antitoxin (YdcD), the functional classification of ydaN is still being investigated. The lack of a clearly defined operon structure or genetic context similar to ydcDE has complicated functional assignment. Sequence homology searches and comparative genomics approaches are recommended for researchers attempting to predict potential functions based on evolutionary relationships.

What are the optimal conditions for expressing recombinant ydaN protein?

For successful expression of recombinant B. subtilis ydaN protein, E. coli expression systems have been effectively employed . The following methodological approach is recommended:

• Expression System Selection: E. coli strains such as BL21(DE3), Rosetta, or SHuffle E are suitable for expression of B. subtilis proteins . For ydaN specifically, standard E. coli expression has been documented .

• Vector Design: Include an N-terminal His-tag for purification purposes. Ensure the construct is verified by DNA sequencing before proceeding with expression .

• Induction Parameters: Typically, IPTG induction at concentrations between 0.1-1.0 mM when culture reaches OD600 of 0.6-0.8, followed by expression at lower temperatures (16-25°C) for 16-20 hours can enhance soluble protein yield.

• Pilot-scale Testing: Conduct small-scale expression trials (25mL culture volume) to optimize conditions before scaling up .

For researchers encountering expression difficulties, alternative approaches using B. subtilis itself as an expression host may be considered, particularly given its strong expression loci and high capacity for enzyme production .

What purification strategy is most effective for obtaining high-purity ydaN protein?

A multi-step purification strategy is recommended for obtaining high-purity recombinant ydaN protein:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or similar matrices is effective for capturing His-tagged ydaN protein .

• Automated Chromatography: Utilize automated ÄKTA chromatography systems for consistent results .

• Buffer Optimization: For storage and stability, a Tris/PBS-based buffer containing 6% trehalose at pH 8.0 has been successfully employed .

• Quality Control: Implement rigorous QC measures including:

  • SDS-PAGE analysis for purity assessment (target >90% purity)

  • Mass spectrometry for sequence verification and post-translational modification identification

  • ELISA if specific antibodies are available

After purification, the protein should be stored according to established protocols: aliquot and store at -20°C/-80°C, with 50% glycerol added as a cryoprotectant to avoid repeated freeze-thaw cycles .

How do expression tags affect ydaN protein function and activity?

The effect of expression tags on protein function varies case by case. For ydaN protein, which is typically expressed with an N-terminal His-tag , the following considerations apply:

• Small Tags Impact: His-tags and similar small tags (such as FLAG) often do not significantly impact protein folding or function. Evidence from structural biology shows more than 100 His-tagged proteins in the Protein Data Bank maintaining their proper folding .

• Activity Assessment: Without established activity assays for the uncharacterized ydaN protein, researchers should design comparative experiments with different tag positions (N-terminal vs. C-terminal) or tag-cleaved versions to assess potential functional impacts.

• Tag Removal Considerations: If activity issues are suspected, incorporation of protease cleavage sites (TEV, PreScission, etc.) between the tag and protein may be beneficial.

• Tag Position Optimization: If N-terminal tagging affects function, C-terminal tagging alternatives should be explored, as terminal accessibility varies between proteins .

For definitive answers regarding tag effects on this specific protein, experimental validation through activity or binding assays is necessary, as computational prediction alone is insufficient.

What are the potential research applications for B. subtilis ydaN protein?

While the specific function of ydaN remains uncharacterized, several research applications can be pursued:

• Functional Genomics Studies: Gene knockout or complementation studies in B. subtilis to determine phenotypic effects and potential physiological roles.

• Protein-Protein Interaction Mapping: Pull-down assays, yeast two-hybrid screens, or proximity labeling approaches to identify interaction partners, which may provide functional insights.

• Cellular Localization Studies: Fluorescent protein fusions or immunolocalization to determine subcellular distribution patterns.

• Structural Biology Investigations: X-ray crystallography or cryo-EM studies to determine three-dimensional structure, potentially revealing functional domains.

• Antimicrobial Research: B. subtilis strains have demonstrated antimicrobial properties against various pathogens, including eye pathogens . Investigating whether ydaN contributes to these properties could be valuable.

• Probiotic Applications: As B. subtilis is increasingly recognized for probiotic applications , understanding the role of ydaN in survival, colonization, or beneficial effects could have translational relevance.

How can I design experiments to elucidate the function of uncharacterized ydaN protein?

A systematic approach to functionally characterize ydaN protein should include:

• Bioinformatic Analysis Pipeline:

  • Sequence homology searches against characterized proteins

  • Domain prediction and conserved motif identification

  • Genomic context analysis (neighboring genes, operon structure)

  • Phylogenetic profiling across bacterial species

• Expression Analysis:

  • Determine conditions under which ydaN is expressed in B. subtilis

  • qRT-PCR or RNA-seq under various stress conditions

  • Promoter-reporter fusion studies to identify regulatory elements

• Genetic Manipulation Experiments:

  • Generate clean deletion mutants and assess phenotypes under various conditions

  • Complementation studies with wild-type and mutated versions

  • Overexpression studies to identify gain-of-function phenotypes

• Biochemical Characterization:

  • In vitro enzymatic activity screening with various substrates

  • Protein interaction studies (pull-downs, crosslinking)

  • Post-translational modification analysis

• Structural Studies:

  • Limited proteolysis to identify stable domains

  • Circular dichroism for secondary structure assessment

  • X-ray crystallography or cryo-EM for detailed structure

This multifaceted approach maximizes the likelihood of functional discovery, as demonstrated with other previously uncharacterized B. subtilis proteins .

What controls should be included when working with recombinant ydaN protein?

Proper experimental controls are essential when working with uncharacterized proteins like ydaN:

• Expression/Purification Controls:

  • Vector-only control (expressing tag alone)

  • Well-characterized B. subtilis protein expressed and purified under identical conditions

  • Denatured protein control to distinguish structure-dependent effects

• Activity Assay Controls:

  • Heat-inactivated protein

  • Related protein with known function (if available)

  • Buffer-only controls to account for buffer components

• Interaction Study Controls:

  • Unrelated protein with same tag to identify tag-mediated interactions

  • Competitive binding controls with untagged protein

  • Negative control pull-downs from cells not expressing potential partners

• Specificity Controls:

  • Site-directed mutants of conserved residues

  • Domain deletion variants

  • Cross-species orthologs to test evolutionary conservation of function

• Protein Quality Controls:

  • Thermal shift assays to confirm proper folding

  • Size exclusion chromatography to verify oligomeric state

  • Mass spectrometry to confirm identity and modifications

How can I address solubility issues when expressing recombinant ydaN protein?

Solubility challenges are common with recombinant proteins. For ydaN protein, consider these approaches:

• Expression Condition Optimization:

  • Lower induction temperature (16-20°C)

  • Reduced inducer concentration

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

• Fusion Partner Strategies:

  • Expression with solubility-enhancing tags (MBP, SUMO, TrxA)

  • Testing different tag positions (N vs C-terminal)

• Buffer Optimization:

  • Screen various pH conditions (pH 6.0-9.0)

  • Test different salt concentrations (100-500 mM NaCl)

  • Addition of stabilizing agents (glycerol, trehalose, arginine)

• Structural Modifications:

  • Express individual domains separately

  • Remove predicted disordered regions

  • Introduce stabilizing mutations based on computational design

• Alternative Expression Systems:

  • Cell-free protein synthesis

  • B. subtilis expression systems which may provide native folding environment

  • Mammalian expression for complex folding requirements

A systematic approach testing these variables will likely yield conditions that improve solubility while maintaining native-like structure and function.

How can I validate potential protein-protein interactions involving ydaN?

Validation of protein-protein interactions requires multiple orthogonal approaches:

• In Vitro Validation Methods:

  • Surface plasmon resonance (SPR) for binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  • Microscale thermophoresis (MST) for affinity determination

  • Analytical ultracentrifugation to characterize complex formation

• Cellular Validation Approaches:

  • Bimolecular fluorescence complementation (BiFC)

  • Förster resonance energy transfer (FRET)

  • Co-immunoprecipitation from native B. subtilis

  • Bacterial two-hybrid systems

• Genetic Validation Strategies:

  • Synthetic genetic interactions between ydaN and partner genes

  • Suppressor screening to identify functional relationships

  • Coordinated expression analysis under various conditions

• Structural Validation:

  • Co-crystallization of ydaN with interaction partners

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

  • Cross-linking mass spectrometry to identify proximity relationships

For each potential interaction, researchers should aim to validate using at least three independent methods before considering the interaction biologically relevant.

How do I address contradictory data when characterizing ydaN function?

When faced with contradictory data in protein characterization:

• Systematic Data Analysis:

  • Create a comprehensive table documenting all experimental conditions

  • Identify variables that differ between contradictory results

  • Apply statistical methods to determine significance of differences

• Experimental Standardization:

  • Ensure protein quality control metrics are consistent

  • Standardize assay conditions and readout methods

  • Implement blinded experimental designs where possible

• Validation Approaches:

  • Deploy orthogonal methods to test the same hypothesis

  • Utilize different expression systems or purification approaches

  • Collaborate with independent laboratories for verification

• Contextual Considerations:

  • Evaluate if contradictions reflect condition-dependent behaviors

  • Consider if post-translational modifications affect results

  • Assess if protein complexes vs. monomeric forms show different activities

The field of contradiction analysis in scientific data suggests employing context validation methods, where multiple document sets are analyzed to detect and resolve conflicting information . This approach can be adapted to experimental data analysis by contextualizing each result within its specific experimental framework.

What bioinformatic tools are most appropriate for analyzing ydaN protein sequence and structure?

For comprehensive analysis of ydaN protein, the following bioinformatic tools are recommended:

Integration of results from multiple tools provides more reliable predictions and can guide experimental design for functional characterization.

How can I determine if ydaN protein has enzymatic activity?

A systematic approach to detecting potential enzymatic activity includes:

• Sequence-Based Prediction:

  • Search for catalytic motifs or active site signatures

  • Compare with known enzyme families

  • Identify conserved residues across homologs

• High-Throughput Screening:

  • Generic enzymatic assays (phosphatase, protease, nuclease activities)

  • Substrate panels based on predicted function

  • Activity-based protein profiling with activity-specific probes

• Metabolite Analysis:

  • Metabolomics comparison between wild-type and ydaN mutants

  • In vitro incubation with cellular extracts followed by mass spectrometry

  • Stable isotope labeling to track potential substrate conversions

• Structure-Guided Approaches:

  • Identify potential active site pockets in structural models

  • Dock potential substrates in silico

  • Design mutations of predicted catalytic residues for validation

• Comparative Analysis:

  • Test activities found in proteins with structural similarity

  • Examine the enzymatic activities in the same protein family

  • Consider potential moonlighting functions based on subcellular location

For example, if investigating nuclease activity similar to EndoA (YdcE) , researchers should design assays with various RNA/DNA substrates and analyze cleavage patterns using gel electrophoresis or fluorescent reporter systems.

What metrics should be used to evaluate the quality of purified recombinant ydaN protein?

Comprehensive quality assessment of purified ydaN should include:

Researchers should establish batch-to-batch consistency parameters and maintain detailed records of purification conditions that yield high-quality protein. For storage, aliquoting in Tris/PBS-based buffer with 6% trehalose at pH 8.0, with addition of 50% glycerol as a cryoprotectant, has been effective for recombinant ydaN protein .

What is the potential role of ydaN in B. subtilis physiology and ecology?

While the specific function of ydaN remains uncharacterized, several hypotheses can be proposed based on B. subtilis biology:

• Stress Response: Many uncharacterized B. subtilis proteins play roles in responding to environmental stresses. YdaN may be involved in adaptation to specific ecological niches or stress conditions.

• Antimicrobial Activity: B. subtilis strains demonstrate antimicrobial activity against various pathogens, including eye pathogens . YdaN could potentially contribute to this capability through direct or indirect mechanisms.

• Signaling or Regulatory Functions: The substantial size of ydaN (680 amino acids) suggests it may have complex domains involved in signaling cascades or regulatory networks.

• Probiotic Properties: As B. subtilis gains recognition as a probiotic organism with spore-forming capabilities that allow survival in harsh environments (including the human gut) , ydaN may contribute to these beneficial properties.

• Specialization Within Microbial Communities: Given B. subtilis' soil habitat and interactions with plants and other microorganisms, ydaN might mediate specific interspecies interactions.

Research directions should include expression profiling under various ecological conditions and comparative genomics across Bacillus species inhabiting different niches to gain context-dependent insights.

How might structural biology approaches advance understanding of ydaN function?

Structural biology provides powerful approaches to uncover functional insights for uncharacterized proteins like ydaN:

• Structure Determination Strategies:

  • X-ray crystallography of full-length protein or functional domains

  • Cryo-electron microscopy for larger assemblies or complexes

  • NMR spectroscopy for dynamic regions or smaller domains

  • Integrative structural biology combining multiple techniques

• Structure-Function Relationships:

  • Identification of conserved structural motifs shared with characterized proteins

  • Mapping of surface properties to predict interaction interfaces

  • Location of potential active sites or ligand-binding pockets

  • Dynamics studies to identify conformational changes

• Methodological Considerations:

  • Construct optimization through limited proteolysis to identify stable domains

  • Co-crystallization with potential binding partners or substrate analogs

  • Use of computational approaches to predict functional sites for mutagenesis

• Applications to ydaN:

  • The substantial size of ydaN (680 amino acids) suggests it likely contains multiple domains

  • Domain-by-domain structural characterization may be more feasible than full-length studies

  • Identification of structural homologs may provide functional hypotheses even with low sequence similarity

Structural information can guide precise mutagenesis experiments, enabling researchers to test specific hypotheses about ydaN function with molecular-level precision.

What emerging technologies could accelerate functional characterization of ydaN?

Several cutting-edge technologies hold promise for elucidating ydaN function:

• CRISPR-Based Approaches:

  • CRISPRi for titratable repression to study dosage effects

  • CRISPR-Cas9 base editing for precise point mutations without selection markers

  • CRISPR activation systems to boost expression in native contexts

• High-Resolution Imaging:

  • Super-resolution microscopy to track subcellular localization

  • Cryo-electron tomography to visualize protein complexes in situ

  • Live-cell single-molecule tracking to study dynamics

• Advanced Proteomics:

  • Proximity labeling (BioID, APEX) to map protein neighborhoods

  • Thermal proteome profiling to identify ligands and substrates

  • Cross-linking mass spectrometry for structural and interaction mapping

• Integrative Omics:

  • Multi-omics approaches combining transcriptomics, proteomics, and metabolomics

  • Network analysis to position ydaN within cellular pathways

  • Temporal profiling during development or stress response

• AI and Machine Learning:

  • Deep learning models trained on protein structure-function relationships

  • Natural language processing of literature for hidden connections

  • Automated hypothesis generation and experimental design

• Synthetic Biology Tools:

  • Biosensors to detect ydaN activity in vivo

  • Minimal cell approaches to define essential interaction partners

  • Engineered B. subtilis strains optimized for protein characterization

Integration of these technologies within a systematic research program would significantly accelerate functional insights into this uncharacterized protein.