Recombinant Staphylococcus aureus UPF0178 protein SAR0734 (SAR0734)

What is Staphylococcus aureus UPF0178 protein SAR0734?

The UPF0178 protein SAR0734 is a protein from Staphylococcus aureus with uncharacterized function (UPF). S. aureus is a common commensal microorganism found in 10-30% of the population but is also responsible for various infections ranging from skin abscesses to severe conditions like endocarditis, pneumonia, and toxic shock syndrome . The UPF0178 family represents proteins with conserved domains whose specific biological functions have not been fully elucidated, making them important targets for basic research.

What expression systems are recommended for recombinant SAR0734 production?

E. coli BL21(DE3) is typically recommended for initial expression trials of S. aureus proteins. This strain carries the λDE3 lysogen that expresses T7 RNA polymerase under IPTG induction, enabling high-level protein expression from T7 promoter-driven plasmids like pET vectors . For SAR0734 expression, common vectors include pET-28a(+) for proteins requiring His-tagging and pET-32a(+) for proteins that may benefit from thioredoxin fusion to improve solubility .

How do I determine the optimal induction conditions for SAR0734?

Optimal induction conditions should be determined empirically through small-scale expression trials varying:

• IPTG concentration (typically 0.1-1.0 mM)

• Induction temperature (15-37°C)

• Induction duration (2-24 hours)

• Cell density at induction time (OD600 0.4-1.0)

Lower temperatures (15-25°C) often promote proper protein folding and solubility, especially for proteins prone to inclusion body formation. Monitor protein expression through SDS-PAGE analysis of samples taken at different time points post-induction .

How does acetate accumulation affect SAR0734 expression in E. coli?

Contrary to common assumptions, research indicates that acetate at concentrations up to 300 mM does not significantly inhibit recombinant protein expression in E. coli BL21(DE3), though it substantially inhibits cell growth . For SAR0734 expression, monitoring acetate levels is important as they may impact:

The key finding is that medium acetate concentration primarily affects cell density rather than the protein expression level per cell .

What strategies can minimize inclusion body formation when expressing SAR0734?

To minimize inclusion body formation:

• Reduce expression rate by lowering temperature (15-25°C) and IPTG concentration

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

• Use fusion tags that enhance solubility (e.g., thioredoxin tag via pET-32a, GST tag)

• Optimize media composition to reduce metabolic burden

• Consider autoinduction media instead of IPTG induction

When testing these strategies, use experimental design approaches with proper controls to systematically evaluate the effect of each variable on protein solubility .

What purification strategy is most effective for recombinant His-tagged SAR0734?

For His-tagged SAR0734, a multi-step purification strategy is recommended:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-based resins

• Intermediate Purification: Ion exchange chromatography based on the protein's theoretical pI

• Polishing: Size exclusion chromatography to remove aggregates and obtain homogeneous protein

Buffer optimization is crucial - consider testing MES buffer (pH 6.5) with moderate salt (300-400 mM NaCl) and 10% glycerol to maintain protein stability during purification, similar to conditions used for other S. aureus proteins .

How should I design experiments to investigate SAR0734 function?

When investigating the function of an uncharacterized protein like SAR0734, implement a systematic experimental design:

• Define clear research questions and hypotheses about potential functions based on:

  • Sequence homology with characterized proteins

  • Structural predictions

  • Genomic context in S. aureus

• Identify appropriate variables:

  • Independent variables: experimental conditions, mutation sites, interaction partners

  • Dependent variables: binding affinity, enzymatic activity, phenotypic changes

  • Control for extraneous variables: temperature, pH, buffer composition

• Use true experimental design with:

  • Control groups (e.g., inactive mutant protein)

  • Experimental groups (wild-type protein or functional mutants)

  • Random distribution of technical replicates

What control experiments are essential when studying SAR0734 interactions with host proteins?

When studying protein-protein interactions:

• Negative controls:

  • Empty vector controls

  • Irrelevant proteins of similar size/properties

  • Heat-denatured SAR0734

• Positive controls:

  • Known interacting protein pairs from S. aureus

  • Tagged control proteins with validated interactions

• Validation through multiple methods:

  • Pull-down assays

  • Surface plasmon resonance

  • Yeast two-hybrid

  • Co-immunoprecipitation from biological samples

Always include proper statistical analysis of interaction data, with at least three biological replicates to ensure reliability .

How do I analyze contradictory results in SAR0734 expression experiments?

When facing contradictory results:

• Systematically evaluate experimental variables:

  • Create a table documenting all variables across experiments (media, strain, induction conditions, etc.)

  • Identify patterns in successful vs. unsuccessful expressions

• Perform controlled comparative experiments:

  • Test multiple expression conditions in parallel

  • Maintain identical handling procedures between samples

• Consider protein-specific factors:

  • Codon usage optimization for E. coli

  • RNA secondary structures affecting translation

  • Post-translational modifications required for function

This systematic approach helps identify critical factors affecting expression outcomes .

How does cellular respiration change during SAR0734 overexpression, and what are the implications?

During recombinant protein overexpression, dissolved oxygen (DO) levels often increase despite constant oxygen supply conditions, indicating reduced cellular respiration . For SAR0734 expression:

• Monitor DO profiles throughout induction:

  • Increasing DO (from ~20% to >60%) suggests metabolic burden

  • Correlate DO changes with protein expression levels and acetate accumulation

• Implications:

  • Metabolic shift from aerobic respiration toward less efficient pathways

  • Reduced capacity for biomass generation

  • Potential limitations in protein folding machinery

• Potential interventions:

  • Fed-batch cultivation with controlled nutrient supply

  • Oxygen uptake rate (OUR) measurements to adjust feeding strategies

  • Consider adjusting inducer concentration to balance expression and metabolic load

What structural analysis approaches are most informative for an uncharacterized protein like SAR0734?

For structural characterization of SAR0734:

• Computational analysis:

  • Homology modeling based on related UPF0178 family proteins

  • Ab initio structure prediction (AlphaFold2, RoseTTAFold)

  • Molecular dynamics simulations to identify flexible regions

• Experimental structure determination:

  • X-ray crystallography (requires optimization of crystallization conditions)

  • NMR spectroscopy (suitable for smaller domains, <25 kDa)

  • Cryo-EM (particularly if SAR0734 forms larger complexes)

• Functional mapping:

  • Limited proteolysis to identify domain boundaries

  • Hydrogen-deuterium exchange mass spectrometry to map protein dynamics

  • Site-directed mutagenesis of conserved residues to correlate structure with function

How might SAR0734 contribute to S. aureus pathogenicity?

S. aureus pathogenicity is multifactorial, involving numerous virulence factors . To investigate SAR0734's potential role:

• Analyze transcriptional regulation:

  • Determine if SAR0734 expression changes during infection processes

  • Identify regulatory elements controlling expression (e.g., stress response, quorum sensing)

• Generate and characterize knockout mutants:

  • Create ΔSar0734 strain and assess virulence in infection models

  • Complement with wild-type or mutant variants to confirm specificity

• Investigate host interactions:

  • Determine if SAR0734 interacts with host immune components

  • Assess impact on biofilm formation or persistence

  • Examine contribution to antibiotic resistance mechanisms

Systematic comparison of wild-type and mutant strains across multiple infection models is crucial for understanding pathogenicity contributions .

What methodological approaches can determine if SAR0734 is surface-exposed or secreted?

To determine cellular localization:

• Bioinformatic prediction:

  • SignalP for signal peptide prediction

  • TMHMM for transmembrane domains

  • SecretomeP for non-classical secretion

  • Analysis of sortase recognition motifs (LPXTG) for cell wall anchoring

• Subcellular fractionation:

  • Separate cellular compartments (cytoplasm, membrane, cell wall, secreted)

  • Western blot using anti-SAR0734 antibodies on each fraction

  • Include controls for each compartment (e.g., sortase A for membrane)

• Fluorescence microscopy:

  • Create fluorescent protein fusions (N- and C-terminal)

  • Immunofluorescence with anti-SAR0734 antibodies

  • Co-localization with known compartment markers

How conserved is SAR0734 across Staphylococcal species and what does this suggest about its function?

To assess evolutionary conservation:

• Conduct comprehensive sequence analysis:

  • BLAST searches against staphylococcal genomes

  • Multiple sequence alignment of homologs

  • Phylogenetic tree construction to visualize relationships

• Analyze synteny:

  • Examine genomic context conservation across species

  • Identify co-evolved genes that may functionally interact with SAR0734

• Identify conserved domains and motifs:

  • Map conservation onto structural models

  • Distinguish highly conserved regions (potential functional sites) from variable regions

Greater conservation typically suggests fundamental biological importance, while strain-specific variations may indicate adaptive functions related to specific niches or pathogenicity mechanisms.

What experimental approaches can determine if SAR0734 interacts with host immune components?

To investigate immune interactions:

• Protein-protein interaction studies:

  • Pull-down assays with human serum components

  • Surface plasmon resonance with purified immune factors

  • ELISA-based binding assays

• Ex vivo assays:

  • Neutrophil activation assays

  • Macrophage phagocytosis experiments

  • Complement activation studies

• In vitro immunomodulation assessment:

  • Cytokine expression profiling in response to purified SAR0734

  • Neutrophil extracellular trap (NET) formation assays

  • Dendritic cell maturation and antigen presentation studies

Experimental conditions should model physiologically relevant parameters (pH, ion concentrations, presence of serum factors) to accurately reflect in vivo interactions .