Recombinant Staphylococcus aureus UPF0403 protein SAR1592 (SAR1592)

What expression systems are most effective for recombinant SAR1592 protein production?

Based on successful approaches with other Staphylococcus aureus proteins, Escherichia coli BL21(DE3)pLysS represents an optimal expression system for recombinant SAR1592 . This strain contains the T7 RNA polymerase gene under control of the lacUV5 promoter and includes the pLysS plasmid to reduce basal expression and mitigate potential toxicity issues.

For recombinant SAR1592 expression, the following methodological workflow is recommended:

• Clone the SAR1592 gene into an expression vector such as modified pMAL-c2

• Include appropriate restriction sites (e.g., NdeI and BamHI) for directional cloning

• Transform the recombinant plasmid into E. coli BL21(DE3)pLysS

• Induce protein expression with IPTG when cultures reach mid-log phase (OD650 ~0.7)

• Continue expression for 4 hours at 37°C before harvesting cells

For proteins with solubility challenges, consider alternative approaches such as:

• Lower induction temperatures (16-20°C)

• Reduced IPTG concentrations (0.1-0.5 mM)

• Fusion with solubility-enhancing tags such as MBP or SUMO

How can I optimize purification of SAR1592 to ensure high yield and purity?

Effective purification of SAR1592 can be achieved using a multi-step chromatography approach similar to that employed for other S. aureus regulatory proteins . The recommended purification protocol includes:

• Initial capture using affinity chromatography:

  • For MBP-tagged constructs, use amylose resin

  • For His-tagged constructs, use Ni-NTA or IMAC resins

  • Include protease inhibitors in initial lysis buffers to prevent degradation

• Intermediate purification:

  • Ion exchange chromatography based on predicted isoelectric point

  • Anion exchange (Q-Sepharose) for acidic proteins

  • Cation exchange (SP-Sepharose) for basic proteins

• Final polishing:

  • Size exclusion chromatography to achieve highest purity

  • Buffer optimization to maintain protein stability

  • Consider tag removal if necessary for downstream applications

Buffer composition significantly impacts stability and yield. Consider the following parameters:

• pH range (typically 7.0-8.0 for S. aureus proteins)

• Salt concentration (150-300 mM NaCl)

• Stabilizing additives (5-10% glycerol, 1-5 mM DTT or TCEP)

What crystallization methods are most successful for S. aureus regulatory proteins like SAR1592?

Based on successful crystallization of similar S. aureus proteins, such as SarR , the following crystallization approach is recommended for SAR1592:

• Initial screening:

  • Employ commercial sparse matrix screens at multiple protein concentrations (5-20 mg/mL)

  • Test both hanging drop and sitting drop vapor diffusion methods

  • Incubate at different temperatures (4°C and 20°C)

  • Evaluate results after 1, 3, 7, and 14 days

• Optimization strategies:

  • Fine-tune promising conditions by varying precipitant concentration in 2% increments

  • Adjust pH in 0.2-0.5 unit increments around initial hits

  • Screen additives (e.g., divalent cations, polyamines, detergents)

  • Implement seeding techniques for poorly nucleating conditions

• Crystal handling and data collection:

  • Develop appropriate cryoprotection protocols (typically 20-25% glycerol, ethylene glycol, or PEG 400)

  • Test diffraction quality on in-house sources before synchrotron data collection

  • Collect complete datasets with appropriate redundancy

The following table summarizes crystallization conditions that have proven successful for S. aureus regulatory proteins:

How should I design experiments to determine if SAR1592 undergoes phosphorylation?

To investigate potential phosphorylation of SAR1592, design experiments based on approaches used for other S. aureus regulatory proteins such as SaeR :

• Bioinformatic analysis:

  • Identify conserved phosphorylation motifs through sequence alignment

  • Focus on aspartic acid residues in the receiver domain, which are likely phosphorylation targets

  • Predict potential phosphorylation sites using structure-based algorithms

• Site-directed mutagenesis:

  • Generate single amino acid substitutions at conserved aspartic acid residues

  • Create aspartic acid to alanine mutations to abolish phosphorylation

  • Include control mutations at non-conserved residues

  • Verify mutations through DNA sequencing and confirm no additional mutations are introduced

• Phosphorylation assays:

  • In vitro phosphorylation using radiolabeled ATP

  • Phosphoprotein-specific staining methods

  • Phos-tag SDS-PAGE for mobility shift detection

  • Mass spectrometry for precise phosphorylation site mapping

• Functional validation:

  • Compare DNA-binding activity between wild-type and phosphorylation-site mutants

  • Assess protein-protein interactions dependent on phosphorylation state

  • Evaluate transcriptional activation capabilities in reporter systems

The methodological approach used for SaeR demonstrated that aspartic acid residue 51 was essential for function , providing a template for similar studies with SAR1592.

What methods are most effective for characterizing DNA-binding properties of SAR1592?

To characterize the DNA-binding properties of SAR1592, employ a comprehensive approach combining in vitro and in vivo methods:

• Electrophoretic Mobility Shift Assay (EMSA):

  • Generate fluorescently labeled or radiolabeled DNA fragments

  • Incubate purified SAR1592 with labeled DNA at various protein:DNA ratios

  • Analyze binding through native gel electrophoresis

  • Include competition with unlabeled DNA to assess specificity

• DNase I footprinting:

  • Identify specific nucleotides protected by SAR1592 binding

  • Map precise binding sites with nucleotide resolution

  • Compare footprints at different protein concentrations

• Chromatin Immunoprecipitation (ChIP):

  • Generate specific antibodies against SAR1592 or use epitope-tagged versions

  • Perform ChIP under various growth conditions to identify in vivo binding sites

  • Couple with next-generation sequencing (ChIP-seq) for genome-wide binding analysis

• Quantitative binding analysis:

  • Surface Plasmon Resonance (SPR) for kinetic and affinity measurements

  • Isothermal Titration Calorimetry (ITC) for thermodynamic parameters

  • Fluorescence Anisotropy for solution-based binding studies

When interpreting DNA-binding data for SAR1592, consider the following experimental variables:

• Buffer composition (particularly salt concentration and pH)

• Presence of divalent cations (Mg²⁺, Ca²⁺)

• Protein concentration and oligomerization state

• Potential effects of phosphorylation on binding affinity and specificity

How can I determine if SAR1592 interacts with other S. aureus proteins in regulatory networks?

To identify and characterize protein-protein interactions involving SAR1592, implement the following methodological approach:

• Affinity-based interaction identification:

  • Affinity purification coupled with mass spectrometry (AP-MS)

  • Express epitope-tagged SAR1592 in S. aureus

  • Purify under mild conditions to maintain interactions

  • Identify co-purifying proteins by mass spectrometry

  • Validate interactions through reciprocal pull-downs

• Direct interaction analysis:

  • Bacterial two-hybrid assays for in vivo interaction detection

  • In vitro pull-down assays with purified candidate partners

  • Surface Plasmon Resonance or Bio-Layer Interferometry for quantitative binding parameters

  • Förster Resonance Energy Transfer (FRET) for interaction dynamics

• Structural characterization of complexes:

  • Co-crystallization with interaction partners

  • Crosslinking mass spectrometry to identify interaction interfaces

  • Hydrogen-deuterium exchange mass spectrometry to map binding regions

• Functional validation of interactions:

  • Mutagenesis of predicted interaction interfaces

  • Competition assays with peptide mimics

  • Evaluation of functional consequences in relevant reporter systems

How should I design experiments to assess the role of SAR1592 in S. aureus virulence?

To investigate the role of SAR1592 in S. aureus virulence, design experiments following approaches successful with other regulatory proteins such as SaeR :

• Generation of isogenic mutants:

  • Create a complete deletion mutant of SAR1592

  • Generate point mutations at conserved functional residues

  • Construct complemented strains expressing wild-type SAR1592

  • Verify mutations by sequencing and confirm no growth defects in vitro

• In vitro virulence phenotype assessment:

  • Hemolysis assays on blood agar plates

  • Quantitative hemolytic activity measurements in liquid culture

  • Expression analysis of known virulence factors using qRT-PCR

  • Biofilm formation assays under various conditions

• Host-pathogen interaction models:

  • Neutrophil survival assays

  • Adhesion to and invasion of relevant host cell types

  • Resistance to antimicrobial peptides and oxidative stress

• In vivo infection models:

  • Murine skin and soft tissue infection model following established protocols

  • Measure abscess formation, tissue bacterial burden, and inflammatory responses

  • Monitor weight loss and other systemic parameters

  • Compare wild-type, mutant, and complemented strains

When designing these experiments, include appropriate controls:

• Wild-type parent strain (positive control)

• Known virulence regulator mutant (comparison control)

• Multiple independent mutant clones to rule out secondary mutations

• Complemented strains to confirm phenotype specificity

What controls are essential when analyzing transcriptomic data related to SAR1592 function?

When conducting transcriptomic analysis to understand SAR1592 function, implement these essential controls and methodological considerations:

• Experimental design controls:

  • Include biological replicates (minimum n=3) for statistical power

  • Maintain consistent growth conditions across all samples

  • Harvest RNA at multiple time points or growth phases

  • Include technical replicates for RNA extraction and library preparation

• Sample preparation quality controls:

  • Verify RNA integrity using Bioanalyzer (RIN > 8.0)

  • Confirm absence of DNA contamination

  • Quantify RNA using multiple methods (spectrophotometry and fluorometry)

  • Validate successful rRNA depletion before library preparation

• Data analysis controls:

  • Use appropriate normalization methods (RPKM, TPM, or DESeq2)

  • Apply suitable statistical thresholds (adjusted p-value < 0.05)

  • Validate expression changes for selected genes using qRT-PCR

  • Compare findings with published datasets for similar regulators

• Functional validation:

  • Confirm direct regulation using ChIP-seq or similar methods

  • Test predicted binding sites using reporter gene assays

  • Evaluate protein production changes for key targets

  • Assess phenotypic consequences of target gene regulation

The following analysis workflow is recommended:

How can I address protein stability issues when working with recombinant SAR1592?

When encountering stability issues with recombinant SAR1592, implement the following systematic troubleshooting approach:

• Buffer optimization:

  • Screen various buffer compositions:

    • pH range (6.5-8.5 in 0.5 unit increments)

    • Salt concentration (100-500 mM NaCl)

    • Buffer systems (Tris, HEPES, phosphate)

  • Test stabilizing additives:

    • Glycerol (5-20%)

    • Reducing agents (DTT, TCEP, β-mercaptoethanol)

    • Amino acids (arginine, glutamate)

  • Evaluate metal ion effects (EDTA vs. divalent cations)

• Storage condition optimization:

  • Compare stability at different temperatures (4°C, -20°C, -80°C)

  • Assess freeze-thaw effects (add 10% glycerol as cryoprotectant)

  • Test lyophilization with appropriate excipients

  • Monitor stability using activity assays and biophysical methods

• Formulation improvements:

  • Try protein stabilizing compounds (trehalose, sucrose, arginine)

  • Optimize protein concentration (dilute vs. concentrated)

  • Consider adding carrier proteins for very dilute samples

  • Test detergents for hydrophobic proteins (non-ionic, below CMC)

• Stability assessment methods:

  • Thermal shift assays to identify stabilizing conditions

  • Size exclusion chromatography to monitor aggregation

  • Dynamic light scattering for polydispersity analysis

  • Activity assays to correlate stability with function

Apply this decision-making framework when troubleshooting SAR1592 stability issues:

What strategies can resolve non-specific binding in SAR1592 DNA-protein interaction studies?

When encountering non-specific binding in SAR1592 DNA interaction studies, implement these methodological solutions:

• Buffer optimization strategies:

  • Systematically increase salt concentration (50-500 mM NaCl)

  • Add non-specific competitors (poly dI-dC, salmon sperm DNA)

  • Include carrier proteins (BSA) to block non-specific binding

  • Test divalent cation effects (Mg²⁺, Ca²⁺)

• Experimental design modifications:

  • Optimize protein:DNA ratios to minimize non-specific interactions

  • Use shorter, more specific DNA fragments for binding studies

  • Implement competitive binding assays with unlabeled DNA

  • Apply more stringent washing conditions for pull-down experiments

• Alternative techniques to validate interactions:

  • DNase I footprinting to precisely identify binding regions

  • SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to determine consensus sequences

  • Microscale thermophoresis for solution-based binding measurements

  • In vivo reporter assays to confirm biological relevance

• Protein quality considerations:

  • Verify protein folding using circular dichroism

  • Assess protein homogeneity by dynamic light scattering

  • Test fresh protein preparations to avoid degradation effects

  • Evaluate the impact of storage conditions on binding specificity

This systematic approach will help distinguish specific from non-specific interactions and improve the reliability of DNA-binding data for SAR1592.

How should I integrate structural and functional data to develop a comprehensive model of SAR1592's role in S. aureus pathogenesis?

To develop a comprehensive model of SAR1592's role in S. aureus pathogenesis, integrate structural and functional data using the following methodological framework:

• Structure-function correlation:

  • Map functional residues identified through mutagenesis onto structural models

  • Identify conserved domains and compare with homologous proteins of known function

  • Use molecular dynamics simulations to understand protein dynamics

  • Predict interaction interfaces based on surface charge distribution and conservation

• Multi-omics data integration:

  • Correlate transcriptomic changes in SAR1592 mutants with direct binding data

  • Integrate proteomic data to assess post-transcriptional effects

  • Incorporate metabolomic profiles to understand downstream physiological impacts

  • Connect molecular phenotypes with virulence characteristics

• Network analysis:

  • Position SAR1592 within the broader regulatory network of S. aureus

  • Identify co-regulated genes and potential regulatory cascades

  • Map epistatic relationships with other regulatory systems

  • Apply systems biology approaches to model regulatory circuits

• Experimental validation of integrated models:

  • Design targeted experiments to test model predictions

  • Use genetic approaches to validate proposed regulatory pathways

  • Employ quantitative models to predict system behavior under various conditions

  • Refine models iteratively based on new experimental data

This integrated approach will provide a comprehensive understanding of how SAR1592 structure relates to its function and its broader role in S. aureus pathogenesis, similar to the understanding developed for regulatory proteins like SaeR .

How can I analyze contradictory results in SAR1592 functional studies across different S. aureus strains?

When confronting contradictory results in SAR1592 studies across different S. aureus strains, implement this systematic analysis approach:

• Strain-specific variation analysis:

  • Compare SAR1592 sequence across strains to identify polymorphisms

  • Assess genetic background differences that might influence SAR1592 function

  • Examine strain-specific regulatory networks through comparative genomics

  • Create isogenic mutants in multiple strain backgrounds to control variables

• Methodological standardization:

  • Implement identical experimental protocols across all strains

  • Standardize growth conditions, media composition, and growth phase

  • Use the same analytical methods and instruments for all measurements

  • Perform experiments in parallel whenever possible

• Statistical validation:

  • Apply appropriate statistical tests for strain comparisons

  • Implement multifactor analysis to identify interaction effects

  • Increase biological replicates to improve statistical power

  • Consider meta-analysis approaches for data integration

• Mechanistic investigation of differences:

  • Test hypothesis-driven experiments to explain strain variation

  • Examine strain-specific post-translational modifications

  • Investigate strain-dependent protein-protein interactions

  • Assess epigenetic factors that might influence gene expression

• Validation in relevant infection models:

  • Compare strain behavior in multiple infection models

  • Correlate in vitro differences with in vivo outcomes

  • Consider host-pathogen interaction variables

  • Evaluate clinical relevance of observed strain differences

This methodological approach will help reconcile contradictory results and provide a more nuanced understanding of SAR1592 function across the diversity of S. aureus strains.