Recombinant Staphylococcus aureus UPF0133 protein SAR0478 (SAR0478)

What is UPF0133 protein SAR0478 and where is it found in Staphylococcus aureus?

UPF0133 protein SAR0478 is a conserved protein found in Staphylococcus aureus, a gram-positive spherically shaped bacterium commonly found in the upper respiratory tract and on human skin . As part of the UPF (Uncharacterized Protein Family) class, it belongs to a group of proteins with conserved sequences but incompletely characterized functions. SAR0478 is encoded in the S. aureus genome and expressed during various growth phases, with notable detection during stationary phase growth as indicated by proteomic studies .

How is UPF0133 protein SAR0478 expression regulated in S. aureus?

Proteomic analyses indicate that UPF0133 family proteins in S. aureus show differential expression based on growth phase. Specifically, UPF0133 protein SAB0428 (a homolog of SAR0478) was detected in the stationary phase with an emPAI value of 0.81 . This suggests that expression may be regulated by growth phase-dependent mechanisms, possibly in response to nutrient limitation or increased cell density. Transcriptional regulators such as SarA, SarR, or SigB may be involved in this regulation, as these control many stationary phase proteins in S. aureus .

What are the recommended methods for recombinant expression of SAR0478?

For recombinant expression of UPF0133 proteins like SAR0478, several expression systems have been successfully employed:

• E. coli expression system: This is commonly used for recombinant S. aureus proteins due to high yield and ease of culture .

• Yeast expression system: This can provide eukaryotic post-translational modifications if required .

• Baculovirus system: For cases where mammalian-like processing is desired .

The expression protocol typically involves:

• Cloning the SAR0478 gene into an appropriate expression vector

• Transformation into the chosen host system

• Induction of protein expression under optimized conditions

• Purification using affinity chromatography (often with His-tag or other fusion tags)

What are the optimal storage conditions for recombinant SAR0478 protein?

Based on similar recombinant S. aureus proteins, the following storage recommendations apply :

For working solutions, it's advisable to store aliquots at 4°C for up to one week and avoid repeated freeze-thaw cycles that can compromise protein integrity .

What purification strategies yield the highest purity for recombinant SAR0478?

For optimal purification of UPF0133 proteins like SAR0478, a multi-step approach is recommended:

• Initial capture: Immobilized metal affinity chromatography (IMAC) if using His-tagged constructs

• Intermediate purification: Ion exchange chromatography to remove contaminants with different charge properties

• Polishing step: Size exclusion chromatography to achieve >90% purity

Researchers should monitor purity using SDS-PAGE at each purification stage, aiming for at least 85-95% purity for most research applications . The choice of tag (His, GST, etc.) should be determined based on downstream applications and potential interference with protein function.

How can SAR0478 be used in studies of S. aureus pathogenesis?

While the specific role of SAR0478 in pathogenesis isn't fully characterized in the available search results, researchers can employ several approaches to investigate its potential contributions:

• Gene knockout studies: Create SAR0478 deletion mutants and assess changes in virulence in infection models

• Transcriptomic analysis: Compare gene expression patterns between wild-type and mutant strains under infection-relevant conditions

• Protein interaction studies: Identify binding partners that may link SAR0478 to known virulence mechanisms

• Immunological studies: Determine if SAR0478 elicits immune responses during infection

The protein could potentially be incorporated into multi-antigen vaccine formulations if found to be immunogenic, similar to the approach used with other S. aureus proteins in the recombinant five-antigen S. aureus vaccine (rFSAV) .

What is known about the role of UPF0133 proteins in S. aureus oxidative stress response?

UPF0133 proteins may play a role in S. aureus oxidative stress responses, which are critical for bacterial survival within phagocytes and during infection . S. aureus employs multiple mechanisms to counter oxidative stress, including:

• Superoxide dismutase (SOD) and catalase enzymes that directly neutralize reactive oxygen species

• MrgA protein that protects DNA by converting the nucleoid from a fibrous to a clogged state under oxidative stress

• Thioredoxin systems that maintain protein thiols in reduced states

UPF0133 proteins like SAR0478 may function in these pathways, particularly given their expression patterns in stationary phase when oxidative stress increases. Researchers should consider examining SAR0478 mutants for altered sensitivity to oxidative stressors like hydrogen peroxide or neutrophil killing assays.

How does SAR0478 compare to UPF0133 proteins in other bacterial species?

UPF0133 proteins appear to be conserved across various bacterial species, suggesting important functional roles . Comparative analysis should consider:

• Sequence homology: Alignment of SAR0478 with UPF0133 proteins from other species to identify conserved domains and motifs

• Genomic context: Examination of neighboring genes that may suggest functional associations

• Expression patterns: Comparison of when and where these proteins are expressed in different species

• Structural similarities: Assessment of predicted or determined structures

For example, researchers might compare SAR0478 with the UPF0133 protein from Chloroflexus aurantiacus (Chy4000558) to identify conserved features that point to fundamental functions of this protein family.

What role might SAR0478 play in S. aureus biofilm formation and persistence?

S. aureus biofilms represent a significant clinical challenge due to their increased resistance to antibiotics and host immune responses. While direct evidence for SAR0478's role in biofilms is not present in the provided search results, several approaches can investigate this connection:

• Biofilm assays: Compare biofilm formation between wild-type and SAR0478 mutant strains

• Spatially resolved proteomics: Use techniques like laser ablation sample transfer to analyze protein distribution within biofilm structures

• Gene expression analysis: Examine SAR0478 expression levels in planktonic versus biofilm growth states

• Protein localization studies: Determine if SAR0478 is differentially distributed in biofilm cells

Given that certain proteins show altered expression in biofilms, including those involved in oxidative stress responses and metabolism, SAR0478 might contribute to biofilm-specific physiological adaptations .

How might SAR0478 interact with the S. aureus respiratory chain and energy metabolism?

The dissertation research discussed in result highlights the importance of respiratory function and metabolism in S. aureus, particularly under stress conditions. Researchers investigating SAR0478's potential role in these processes should consider:

• Metabolomic analysis: Compare metabolite profiles between wild-type and SAR0478 mutant strains

• Membrane potential assays: Assess if SAR0478 affects membrane potential using fluorescent probes

• Oxygen consumption measurements: Determine if SAR0478 influences respiratory capacity

• Enzymatic activity assays: Test activities of key metabolic enzymes in the presence and absence of SAR0478

The protein might function similarly to other stress-responsive proteins that help modulate metabolic pathways under changing environmental conditions, particularly during the transition to stationary phase when it appears to be expressed .

What is the potential of SAR0478 as a target for novel antimicrobial strategies?

With increasing antibiotic resistance in S. aureus (including MRSA strains), novel targets for antimicrobial development are urgently needed. For researchers exploring SAR0478 as a potential drug target:

• Essentiality assessment: Determine if SAR0478 is essential for S. aureus viability or virulence

• Structural analysis: Identify potential binding pockets that could be targeted by small molecules

• High-throughput screening: Develop assays to identify inhibitors of SAR0478 function

• In silico docking studies: Use computational approaches to screen virtual compound libraries

If SAR0478 proves to be involved in stress responses or virulence, it might represent a novel target for antimicrobial development that could circumvent existing resistance mechanisms in S. aureus.

How can researchers address solubility issues when working with recombinant SAR0478?

Membrane-associated or hydrophobic proteins often present solubility challenges during recombinant expression. Strategies to improve SAR0478 solubility include:

• Optimization of expression conditions:

  • Reduce expression temperature (16-25°C)

  • Use lower inducer concentrations

  • Select appropriate E. coli strains (BL21(DE3)pLysS, Rosetta, etc.)

• Buffer optimization:

  • Include mild detergents (0.05-0.1% Triton X-100, NP-40)

  • Test various salt concentrations (150-500 mM NaCl)

  • Add stabilizing agents like glycerol (5-20%)

• Fusion tags:

  • Consider solubility-enhancing tags (MBP, SUMO, Thioredoxin)

  • Use removable tags with specific protease sites

Based on protocols for similar proteins, researchers should systematically test these approaches to identify optimal conditions for their specific construct .

What are effective approaches for detecting protein-protein interactions involving SAR0478?

To investigate the protein interaction network of SAR0478, researchers can employ multiple complementary techniques:

• Pull-down assays: Using tagged recombinant SAR0478 as bait to capture interaction partners from S. aureus lysates

• Bacterial two-hybrid systems: For detecting direct protein-protein interactions

• Cross-linking coupled with mass spectrometry: To identify transient or weak interactions

• Surface plasmon resonance (SPR): For measuring binding kinetics with purified candidate partners

• Co-immunoprecipitation: Using specific antibodies against SAR0478 or potential partners

These approaches can help map the functional context of SAR0478 within cellular networks and potentially reveal its role in S. aureus physiology .

What controls should be included when studying the function of SAR0478 in S. aureus?

Rigorous experimental design requires appropriate controls when investigating SAR0478 function:

• Genetic controls:

  • Empty vector controls for complementation studies

  • Wild-type strain for comparison with mutants

  • Complemented mutant strain to confirm phenotype specificity

• Protein controls:

  • Heat-inactivated SAR0478 for functional assays

  • Unrelated proteins of similar size for specificity tests

  • Known functional proteins as positive controls for assays

• Experimental controls:

  • Growth condition controls (media, temperature, oxygen availability)

  • Time point controls for growth phase-specific effects

  • Technical replicates to ensure reproducibility

  • Biological replicates to account for strain variation

Implementing these controls will strengthen the validity and reproducibility of findings related to SAR0478 function .

How might SAR0478 contribute to S. aureus adaptation during host-pathogen interactions?

S. aureus is known for its remarkable adaptability to diverse host environments, including survival within professional phagocytes . Future research into SAR0478's role in these adaptations could focus on:

• Expression in infection models: Measure SAR0478 expression levels during various stages of infection

• Host factor responses: Determine if SAR0478 expression changes in response to specific host factors (antimicrobial peptides, nutrient limitation, pH shifts)

• Immune evasion: Investigate if SAR0478 contributes to mechanisms that counter host immune responses

• Co-culture studies: Examine SAR0478 expression in co-culture with host cells or other microorganisms

These approaches may reveal previously unknown functions of SAR0478 in the complex dynamics of host-pathogen interactions .

What techniques are being developed to study the temporal dynamics of SAR0478 expression?

Advanced methods for monitoring protein expression dynamics in real-time can provide insights into when and how SAR0478 functions:

• Reporter gene fusions: Creating SAR0478 promoter-GFP/luciferase fusions to monitor expression patterns

• Time-resolved proteomics: Sampling at multiple time points to track protein abundance changes

• Single-cell analysis: Using fluorescent reporter strains to examine cell-to-cell variation in expression

• Microfluidic devices: Observing expression dynamics under changing environmental conditions

These approaches can reveal the temporal regulation of SAR0478 in response to environmental cues and stressors, providing insights into its biological role .

How can systems biology approaches enhance our understanding of SAR0478 function?

Integrative systems biology approaches can place SAR0478 within the broader context of S. aureus physiology:

• Multi-omics integration: Combining transcriptomics, proteomics, and metabolomics data to build comprehensive models

• Network analysis: Constructing protein interaction networks to identify functional modules involving SAR0478

• Flux balance analysis: Modeling metabolic pathways to predict effects of SAR0478 perturbation

• Machine learning applications: Using AI to identify patterns in large datasets that suggest SAR0478 functions

These computational approaches, combined with experimental validation, can generate testable hypotheses about SAR0478's role in complex cellular processes .