Recombinant Bacillus subtilis UPF0053 protein yhdT (yhdT)

What is the UPF0053 protein yhdT from Bacillus subtilis?

The yhdT protein is an uncharacterized protein belonging to the UPF0053 protein family in Bacillus subtilis. It is encoded by the yhdT gene in B. subtilis strain 168. The protein is relatively small, consisting of 80 amino acids in its full-length form . As an uncharacterized protein, its precise biological function remains to be fully elucidated, making it an interesting target for fundamental research into bacterial protein function and interactions .

What are the basic biochemical properties of the yhdT protein?

The yhdT protein has the following biochemical characteristics:

How can I express recombinant yhdT protein in a laboratory setting?

The recombinant expression of yhdT can be achieved using several systems, with E. coli being the most commonly employed host. Based on current methodologies:

• Vector selection: pET-based expression vectors are commonly used for recombinant protein expression. For yhdT specifically, systems that allow for tag addition (such as His-tag) facilitate subsequent purification .

• Host strain: E. coli strains optimized for protein expression such as BL21(DE3) are suitable for yhdT expression .

• Expression protocol:

  • Transform the expression vector containing the yhdT gene into the host strain

  • Grow transformed cells in appropriate media (typically LB with appropriate antibiotics)

  • Induce protein expression (commonly with IPTG if using a T7-based system)

  • Harvest cells and extract protein using suitable lysis methods

• Alternative expression in B. subtilis: If native-like conditions are desired, B. subtilis WB800N strain can be used as shown in other recombinant protein studies. This strain has reduced protease activity, making it suitable for heterologous protein expression .

What purification strategies yield the highest purity of recombinant yhdT?

For optimal purification of recombinant yhdT protein:

• Affinity chromatography: If expressed with a His-tag, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin is highly effective. The protocol should include:

  • Cell lysis in a buffer containing 20-50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 10 mM imidazole

  • Binding to Ni-NTA resin

  • Washing with increasing imidazole concentrations (20-50 mM)

  • Elution with high imidazole (250-500 mM)

• Secondary purification: For higher purity, follow with size exclusion chromatography (SEC) using a suitable column (Superdex 75 or similar) in a physiological buffer .

• Quality assessment: Purity should be analyzed using SDS-PAGE (>85% purity is typically achievable with this approach) .

• Storage considerations: The purified protein can be stored with 50% glycerol at -20°C/-80°C to maintain stability. For lyophilized preparations, reconstitution in deionized sterile water to 0.1-1.0 mg/mL is recommended .

How can I verify the structural integrity of purified yhdT protein?

To confirm the structural integrity of purified yhdT:

• Western blotting: Using antibodies against the tag or, if available, against the protein itself

• Mass spectrometry:

  • MALDI-TOF or ESI-MS to confirm molecular weight

  • Peptide mass fingerprinting after tryptic digestion for sequence verification

• Circular dichroism (CD) spectroscopy: To assess secondary structure elements and proper folding

• Dynamic light scattering (DLS): To check for aggregation and determine the hydrodynamic radius

• Limited proteolysis: To evaluate domain organization and stability

What are the methodological approaches for studying yhdT protein-protein interactions?

Several techniques can be employed to study yhdT interactions with other proteins:

• Yeast Two-Hybrid (Y2H) screening:

  • Clone yhdT as bait (BD fusion) and screen against a B. subtilis cDNA library

  • Use a matrix-based approach for known candidate interactions

  • Validate positive interactions using in vitro methods

• Tandem Affinity Purification coupled with Mass Spectrometry (TAP-MS):

  • Generate a TAP-tagged yhdT construct

  • Express in B. subtilis for native interactions or in a heterologous system

  • Purify protein complexes using the dual affinity tags

  • Identify interaction partners by mass spectrometry

• Proximity-based labeling:

  • Recent methods developed at the University of Chicago enable detection of proteins in close proximity within individual cells

  • This approach provides information about functional protein groupings rather than just abundance

• Cross-linking studies:

  • Chemical cross-linking followed by mass spectrometry (XL-MS)

  • Photo-crosslinking using genetic code expansion in B. subtilis with non-standard amino acids

How can genetic code expansion techniques be applied to study yhdT function?

Genetic code expansion provides powerful tools for studying yhdT:

• Incorporation of non-standard amino acids (nsAAs):

  • B. subtilis genetic code expansion systems can incorporate up to 20 distinct nsAAs

  • Three different families of genetic code expansion systems and two codon choices are available

  • These systems enable click-labeling, photo-crosslinking, and translational titration

• Methodological approach:

  • Select appropriate orthogonal tRNA/aminoacyl-tRNA synthetase pairs

  • Design constructs with amber (UAG) codons at sites of interest

  • Express in B. subtilis in media supplemented with the desired nsAA

  • Applications include site-specific fluorescent labeling, crosslinking for interaction studies, and photo-control of protein function

• Example protocol for photo-crosslinking:

  • Incorporate photo-reactive nsAAs (e.g., p-benzoyl-L-phenylalanine) at predicted interaction sites

  • UV-irradiate to activate crosslinking

  • Analyze crosslinked products by Western blotting and mass spectrometry

How can I optimize expression of recombinant yhdT in Bacillus subtilis?

To optimize yhdT expression in B. subtilis:

• Promoter selection:

  • For constitutive expression: P43 or Pveg promoters

  • For inducible expression: IPTG-inducible Pgrac212 system or xylose-inducible PxylA system

  • For self-inducing systems: Psrfa-based systems that respond to quorum sensing

• Strain selection:

  • B. subtilis WB800N has eight deleted protease genes, improving protein yield

  • For secreted expression, consider strains with enhanced secretion capacity

• Codon optimization:

  • Adapt the coding sequence to B. subtilis codon usage bias

  • Remove rare codons and optimize GC content

• Expression enhancement strategies:

  • Co-expression with chaperones to improve folding

  • Optimization of culture conditions (temperature, media composition)

  • Signal peptide selection for secreted expression

What approaches can be used to determine the functional role of yhdT protein?

To investigate the function of this uncharacterized protein:

• Gene knockout and phenotypic analysis:

  • Generate a yhdT deletion strain in B. subtilis

  • Compare growth under various conditions (different temperatures, pH, osmotic stress)

  • Examine cell morphology, division, and stress responses

• Transcriptomic and proteomic analysis:

  • RNA-Seq of wild-type vs. ΔyhdT strains

  • Proteome analysis using methods similar to those in the UK Biobank proteomic study

  • Metabolomic profiling to identify affected pathways

• Genome-scale metabolic modeling:

  • Utilize systems biology approaches such as the B. subtilis ME-model (iJT964-ME)

  • Predict potential metabolic roles and stress responses

  • Validate predictions experimentally

• Localization studies:

  • GFP fusion proteins to determine subcellular localization

  • Membrane fractionation to confirm membrane association

  • Immunogold electron microscopy for high-resolution localization

How does yhdT expression respond to different stress conditions in B. subtilis?

Understanding stress responses requires:

• Quantitative expression analysis:

  • RT-qPCR to measure transcript levels under different stresses

  • Western blotting with specific antibodies to monitor protein levels

  • Reporter gene fusions (e.g., yhdT promoter-GFP) for real-time monitoring

• Stress conditions to test:

  • Ethanol stress (relevant to B. subtilis stress response mechanisms)

  • Salt stress

  • Oxidative stress (H₂O₂ or paraquat)

  • Nutrient limitation

  • Heat shock

• Integration with omics data:

  • Compare with datasets from the ME-model validation studies

  • Correlate yhdT expression changes with global stress response patterns

What protein quantification methods are most accurate for determining yhdT concentration?

For accurate quantification of yhdT protein:

• Direct methods:

  • Amino acid analysis (AAA): The gold standard method that determines actual protein content by measuring individual amino acids after hydrolysis

  • Spectrophotometric methods using calculated extinction coefficients

• Indirect methods and their limitations:

  • Bradford assay: Prone to interference from detergents and buffer components

  • Lowry method: More sensitive than Bradford but influenced by non-protein substances

  • BCA assay: Compatible with many detergents but affected by reducing agents

• Comparison of methods:

Amino acid analysis is recommended as the most accurate method for yhdT quantification in research settings .

How can I design experiments to validate predicted protein-protein binding interfaces involving yhdT?

To validate predicted interaction interfaces:

• Computational prediction of interfaces:

  • Use protein-protein docking software

  • Predict critical residues at the interface

  • Generate testable hypotheses about interaction mechanisms

• Mutational analysis:

  • Create alanine scanning mutations at predicted interface residues

  • Express and purify mutant proteins

  • Measure binding affinities using biophysical methods

• Photo-crosslinking validation:

  • Incorporate photo-reactive amino acids at predicted interface sites using genetic code expansion in B. subtilis

  • Perform crosslinking experiments to capture interactions

  • Identify crosslinked peptides by mass spectrometry

• Biophysical validation methods:

  • Surface Plasmon Resonance (SPR) to measure binding kinetics

  • Isothermal Titration Calorimetry (ITC) for thermodynamics of binding

  • NMR chemical shift perturbation to map interaction surfaces

This approach has proven successful in validating predicted protein-protein binding interfaces in B. subtilis, as demonstrated in recent genetic code expansion studies .

How can metabolic modeling approaches improve our understanding of yhdT function?

Metabolic modeling provides valuable insights into yhdT function:

What emerging technologies show promise for studying uncharacterized proteins like yhdT?

Several cutting-edge approaches are particularly valuable for studying proteins with unknown functions:

• AI-driven protein design and analysis:

  • New AI frameworks incorporate experimental data and text-based narratives to accelerate protein research

  • These approaches can generate testable hypotheses about protein function based on structural features

  • A combination of simulation data and experimental validation can optimize protein design and function prediction

• High-throughput protein interaction mapping:

  • Recent advances in protein proximity labeling enable mapping of protein interaction networks at unprecedented scale

  • These methods provide insights into functional relationships and can suggest roles for uncharacterized proteins

• Single-cell proteomics:

  • New methods for studying proteins at the single-cell level can reveal cell-to-cell variability in protein abundance and localization

  • This approach is particularly valuable for understanding heterogeneous responses to stress conditions

• CRISPR-based functional genomics:

  • Systematic gene editing and screening approaches can reveal genetic interactions

  • CRISPRi/CRISPRa systems allow controlled modulation of gene expression without permanent genetic changes

  • These approaches can help position yhdT within cellular pathways

What is the recommended experimental workflow for characterizing yhdT function?

Based on current methodologies, we recommend the following research workflow:

• Initial characterization:

  • Expression and purification of recombinant yhdT protein

  • Structural analysis (secondary structure prediction, disorder analysis)

  • Subcellular localization studies

• Functional analysis:

  • Gene knockout and phenotypic characterization

  • Global approaches (transcriptomics, proteomics) to identify affected pathways

  • Protein-protein interaction studies using multiple complementary techniques

• Mechanistic studies:

  • Site-directed mutagenesis of conserved residues

  • Genetic code expansion for photo-crosslinking

  • Detailed biochemical characterization of any identified activities

• Integration with systems biology:

  • Incorporation of findings into metabolic models

  • Generation and testing of new hypotheses from model predictions

  • Iterative refinement of functional understanding

This comprehensive workflow leverages cutting-edge techniques while maintaining rigorous validation at each step to ensure reliable characterization of this uncharacterized protein .