Recombinant Staphylococcus aureus UPF0337 protein SAR0874 (SAR0874)

What is Recombinant Staphylococcus aureus UPF0337 protein SAR0874?

Recombinant Staphylococcus aureus UPF0337 protein SAR0874 is a small protein belonging to the CsbD family of proteins, with a molecular weight of approximately 7,019 Da. The protein consists of 64 amino acids with the sequence MADESKFEQAKGNVKETVGNVTDNKNLENEGKEDKASGKAKEFVENAKEKATDFIDKVKGNKGE as identified in UniProt (Q6GIH6) . SAR0874 is classified as an UPF0337 protein, which indicates it belongs to a family of uncharacterized protein functions, suggesting its complete functional characterization remains an active area of investigation. The protein is found in S. aureus strain MRSA252, a clinically significant methicillin-resistant strain, indicating its potential relevance to pathogenicity or antibiotic resistance mechanisms . As a CsbD family protein, it shares homology with stress-inducible proteins in other bacterial species, suggesting possible roles in stress response pathways.

How can I confirm the identity and purity of SAR0874 protein samples?

Confirming the identity and purity of SAR0874 protein samples requires a multi-analytical approach. SDS-PAGE analysis should reveal a single band at approximately 7 kDa, with purity standards for research applications being ≥85% as determined by this method . Western blotting using anti-SAR0874 antibodies provides identity confirmation, while mass spectrometry should verify the exact molecular weight of 7,019 Da (±0.1%). N-terminal sequencing can confirm the first 10-15 amino acids match the expected sequence. For higher confidence in structural integrity, circular dichroism spectroscopy can provide a spectral profile consistent with the protein family's secondary structure characteristics.

For recombinant versions of the protein, be aware that tag additions (N-terminal or C-terminal) will alter the molecular weight and potentially the migration pattern on gels. Peptide mass fingerprinting after tryptic digestion, comparing the resultant peptide masses with in silico predictions based on the known sequence, offers additional verification of protein identity. For functional studies requiring high confidence in protein quality, combining multiple analytical techniques is essential to establish both identity and structural integrity.

What expression systems are optimal for producing SAR0874 protein?

Multiple expression systems can be utilized for SAR0874 production, each with distinct advantages depending on research requirements:

For basic structural and preliminary functional studies, E. coli-based expression is typically sufficient for SAR0874 due to its relatively small size and bacterial origin . The BL21(DE3) strain with pET vector systems offers efficient expression. For optimal results, consider using codon-optimized sequences for the expression host and include a cleavable affinity tag (His6 or GST) to facilitate purification while allowing tag removal for downstream applications. Expression optimization should include testing various induction temperatures (16-37°C), IPTG concentrations (0.1-1.0 mM), and induction durations (3-24 hours) to maximize soluble protein yield.

What storage conditions are recommended for maintaining SAR0874 protein stability?

Optimal storage conditions for SAR0874 protein depend on the intended duration of storage and subsequent applications. For short-term storage (1-7 days), maintaining the protein at 4°C in an appropriate buffer with protease inhibitors is suitable. For medium-term storage (1-4 weeks), keeping the protein at -20°C in aliquots with 10-20% glycerol as a cryoprotectant is recommended. Long-term storage (months to years) requires either -80°C storage or lyophilization to maintain stability .

The buffer composition significantly impacts stability. For SAR0874, a standard buffer containing 20-50 mM Tris-HCl or phosphate buffer (pH 7.0-8.0), 100-150 mM NaCl, and 1-5 mM DTT or 2-ME as reducing agents is typically appropriate. Adding 10% glycerol can enhance stability. Repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and aggregation. For working aliquots stored at 4°C, protease inhibitors should be included to prevent degradation. Stability should be periodically assessed using SDS-PAGE and functional assays appropriate to your research questions.

What are the known functional roles of SAR0874 in Staphylococcus aureus?

SAR0874, as a member of the CsbD family of proteins, is believed to play roles in stress response mechanisms in Staphylococcus aureus, though its precise functions remain under investigation. Current research suggests several potential functions, including stress response based on homology to stress-inducible proteins, metabolic regulation indicated by transcriptome alterations in knockout studies, and possible contributions to virulence pathways .

Research indicates that SAR0874, like other CsbD family proteins, may be involved in bacterial adaptation to environmental stressors. Genomic analysis places it in the context of genes related to oxidative stress responses and general stress adaptation. Transcriptomic studies have shown differential expression under various stress conditions, suggesting a regulatory role in metabolic adaptation. There is emerging evidence for its potential association with respiratory metabolism and oxidative stress responses. While direct experimental evidence for specific biochemical functions remains limited, ongoing research using knockout mutants and comparative genomics approaches continues to elucidate its physiological significance in S. aureus survival and pathogenicity.

How does SAR0874 contribute to S. aureus virulence?

The contribution of SAR0874 to S. aureus virulence is an active area of research, with several lines of evidence suggesting potential roles in stress adaptation during infection, oxidative stress resistance, metabolic adaptation in host environments, and possibly immune evasion mechanisms. Research on S. aureus virulence factors has identified connections between stress response proteins and pathogenicity . SAR0874, being associated with the stress response network, potentially contributes to bacterial survival during the transition from commensal to pathogenic states.

Studies have indicated potential relationships between SAR0874 and pathways involved in oxidative stress resistance, which is crucial for S. aureus survival during neutrophil attack. Research approaches to investigate these connections include RNA-seq and RT-qPCR during infection models, oxidative stress challenge assays, NO sensitivity testing, metabolomics, and isotope labeling studies. To definitively establish its role in virulence, researchers should consider gene knockout studies followed by virulence assessment in appropriate animal models, along with complementation studies to confirm observed phenotypes are directly attributable to SAR0874 function.

What experimental approaches are suitable for studying SAR0874 protein interactions?

Multiple complementary approaches can be employed to study SAR0874 protein interactions. Pull-down assays provide initial screening of interacting partners but require tagged SAR0874, which may introduce artifacts. Co-immunoprecipitation validates interactions in native conditions but depends on antibody specificity and availability. Yeast two-hybrid and bacterial two-hybrid systems offer systematic screening of protein-protein interactions, with bacterial two-hybrid being better suited for bacterial proteins like SAR0874. Surface plasmon resonance provides quantitative interaction kinetics but requires purified interacting partners. Cross-linking mass spectrometry can identify interaction interfaces but involves complex data analysis .

When designing interaction studies for SAR0874, consider both its small size (64 amino acids) and potential functional domains. The protein's compact structure may limit interaction surface availability, necessitating careful tag placement to avoid interfering with binding sites. For pull-down experiments, both N-terminal and C-terminal tagged versions should be tested to identify potential interference with interaction capabilities. Validation of interactions should employ multiple methodologies, ideally including at least one approach that examines interactions in the native cellular environment (e.g., in vivo cross-linking).

How does SAR0874 relate to bacterial stress responses?

SAR0874, as a member of the CsbD protein family, is implicated in bacterial stress response mechanisms. Research suggests potential roles in protection against oxidative stress, interaction with nitrosative stress pathways, adaptation to nutrient limitation, and possible contributions to antibiotic tolerance . Studies indicate that SAR0874 may function in coordination with the bacterial nitric oxide synthase (NOS) system, potentially influencing endogenous oxidative stress levels and respiratory function.

Transcriptomic analyses have revealed altered expression of SAR0874 under various stress conditions, including exposure to reactive oxygen species, nutrient deprivation, and antibiotic challenge. The protein's exact biochemical mechanism remains to be fully characterized, but its conservation across Staphylococcal species suggests an important role in adaptation to stressful environments. Experimental approaches to further elucidate its function should include phenotypic characterization of deletion mutants under various stress conditions, complementation studies, and identification of genetic and physical interactions with known stress response pathways.

What are the challenges in studying structure-function relationships of SAR0874?

Investigating the structure-function relationships of SAR0874 presents several challenges, primarily related to its small size of only 64 amino acids. This limited size constrains the number of structural domains and makes crystallization challenging. The protein shows limited homology to well-characterized proteins, making it difficult to predict function from sequence alone. SAR0874 may undergo dynamic conformational changes, with function potentially depending on structural flexibility. Additionally, its functionality may be interaction-dependent, with structure altering upon binding to partners .

The small size of SAR0874 (7,019 Da) makes X-ray crystallography difficult, though Nuclear Magnetic Resonance (NMR) spectroscopy represents a more viable approach for such small proteins. Computational approaches, including homology modeling and molecular dynamics simulations, can provide preliminary structural insights, particularly if structures of homologous CsbD family proteins are available. For complete structure-function analysis, integrating structural data with site-directed mutagenesis of conserved residues, followed by functional assays, can correlate specific structural elements with biological activities. Structural studies under varying conditions may reveal functionally relevant conformational changes.

How can I design experiments to investigate SAR0874's role in metabolic pathways?

Designing comprehensive experiments to elucidate SAR0874's role in metabolic pathways requires a multi-faceted approach. Gene deletion and complementation studies establish baseline phenotypes and should use clean deletion methods such as allelic exchange. Transcriptomics through RNA-seq can identify affected pathways by comparing multiple conditions relevant to infection. Metabolomics detects metabolic profile changes by targeting both central and peripheral metabolism. Isotope labeling studies track specific metabolic fluxes using isotopes selected based on hypothesized pathways. Protein-protein interaction studies identify metabolic enzyme interactions, while biochemical enzyme assays measure specific enzymatic activities by comparing wildtype and mutant backgrounds .

When designing these experiments, adopt a systematic approach starting with phenotypic characterization of SAR0874 deletion mutants under various metabolic conditions. Use global approaches like RNA-seq and metabolomics to identify affected pathways, followed by targeted validation. For metabolomics studies, analyze both intracellular and secreted metabolites to capture comprehensive metabolic changes. Consider using 13C-labeled carbon sources to track carbon flux through central metabolic pathways. To establish direct versus indirect effects, complement deletion mutants with controlled expression systems allowing titration of SAR0874 levels.

What are the best approaches for studying SAR0874 in the context of antimicrobial resistance?

Investigating SAR0874's potential role in antimicrobial resistance requires integrating multiple research strategies. Susceptibility testing determines MIC changes in deletion mutants across multiple antibiotic classes. Transcriptomics during antibiotic exposure identifies co-regulated resistance genes through time-course analysis of adaptation responses. Overexpression studies assess direct contribution to resistance using inducible expression systems. Biofilm formation assays evaluate contribution to tolerance mechanisms by comparing planktonic versus biofilm resistance. Epistasis analysis examines interaction with known resistance mechanisms through double mutants with established resistance genes .

Design your investigations to distinguish between direct resistance mechanisms (where SAR0874 directly interferes with antibiotic action) and indirect tolerance mechanisms (where SAR0874 contributes to stress adaptation or metabolic alterations that enhance survival). Include both conventional antibiotics and host-derived antimicrobial peptides in susceptibility testing. Consider the possible connection between SAR0874 and stress response pathways, particularly those related to oxidative stress, which can influence resistance to several antibiotic classes. Time-course experiments examining gene expression and protein levels following antibiotic exposure can reveal whether SAR0874 participates in immediate resistance or adaptive tolerance.

How can I analyze SAR0874 expression under different environmental conditions?

Comprehensive analysis of SAR0874 expression requires strategic experimental design across multiple conditions. RT-qPCR provides quantitative expression measurement but requires validated reference genes. Western blotting verifies protein-level expression but depends on antibody availability. Transcriptomics through RNA-seq provides global expression context including pathway co-regulation information. Reporter fusions allow dynamic monitoring of expression using GFP or luciferase for live monitoring. Proteomics assesses protein abundance in cellular context through MS-based quantification. Single-cell analysis reveals expression heterogeneity using flow cytometry or microscopy with reporters .

When designing expression studies, consider physiologically relevant conditions that S. aureus encounters during infection and colonization. These should include variations in oxygen availability, nutrient availability, pH changes, osmotic stress, and exposure to host factors. Time-course analyses during growth phase transitions can reveal potential roles in adaptation to changing population densities. For reporter fusion studies, ensure the promoter region includes all potential regulatory elements and validate that the reporter construct does not interfere with normal regulation. Integration of expression data with phenotypic observations and metabolic analyses can provide a comprehensive understanding of SAR0874's role in S. aureus biology.

What are common issues in SAR0874 protein purification and how can they be resolved?

Several challenges may arise during SAR0874 purification, each requiring specific troubleshooting approaches. Low expression yield may result from codon bias or protein toxicity, addressed through codon optimization, use of specialized expression strains, or lower induction temperature. Insoluble protein or inclusion body formation indicates improper folding or high expression rates, resolved by reducing induction temperature, co-expressing chaperones, or using solubility tags. Protein degradation may result from protease activity or protein instability, mitigated by adding protease inhibitors, optimizing buffer conditions, and reducing purification time .

For SAR0874, its small size (7,019 Da) presents unique challenges. If encountering solubility issues, consider fusion tags that enhance solubility (such as MBP, SUMO, or Thioredoxin) rather than simple affinity tags. To address potential degradation, conduct purification at 4°C and include protease inhibitors in all buffers. Size exclusion chromatography can be particularly effective as a polishing step to separate the target protein from higher molecular weight contaminants. If the protein forms aggregates, screen various buffer conditions systematically, varying pH (6.0-9.0), salt concentration (50-500 mM NaCl), and additives (glycerol, reducing agents).

How can I optimize antibody-based detection of SAR0874?

Optimizing antibody-based detection of SAR0874 requires consideration of several factors. Western blotting must address transfer efficiency challenges for small proteins by using PVDF membranes and optimized transfer conditions. Immunofluorescence needs to account for fixation impact on epitope accessibility by testing multiple fixation methods. ELISA optimization should compare direct versus sandwich approaches based on antigen coating efficiency. Immunoprecipitation must ensure antibody binding under native conditions through crosslinking approaches and optimized lysis buffers .

The small size of SAR0874 (7,019 Da) presents specific challenges for antibody detection. For Western blotting, use high percentage gels (15-20%) to resolve the protein effectively, and consider tricine-SDS-PAGE systems for better resolution of small proteins. When generating antibodies against SAR0874, select immunogenic epitopes carefully, potentially focusing on regions with predicted surface exposure based on structural models. For all applications, include appropriate controls: recombinant SAR0874 as a positive control and samples from SAR0874 deletion mutants as negative controls. When optimizing protocols, systematically vary antibody concentrations, incubation times/temperatures, and blocking agents to identify optimal conditions.

What controls should be included in experiments involving SAR0874?

Robust experimental design for SAR0874 studies requires comprehensive controls across different experiment types. Expression analysis should include housekeeping gene controls and no-template controls for normalization and contamination detection. Protein interaction studies need non-specific binding controls and tag-only controls to distinguish specific from non-specific interactions. Functional assays must compare wildtype, knockout, and complemented strains to establish causality of observed phenotypes .

How can I address data inconsistencies in SAR0874 functional studies?

Addressing inconsistencies in SAR0874 functional studies requires systematic troubleshooting of various potential sources. Strain background variation may result from genetic differences between laboratory strains, addressed through using isogenic strains and sequence verification. Growth condition variability arises from subtle differences in media, temperature, or oxygenation, requiring standardized protocols and careful monitoring of growth parameters. Genetic compensation involves activation of alternate pathways in deletion mutants, addressed through acute depletion studies and temporal analyses .

When confronting inconsistent results, first verify the genetic integrity of your strains through whole-genome sequencing or targeted verification of the SAR0874 locus and relevant regulatory regions. Consider whether strain background differences might influence results; S. aureus exhibits significant strain-to-strain variation that can impact functional studies. Standardize growth conditions meticulously, including media preparation, culture vessel types, and growth phase at harvesting. When deletion mutants show inconsistent phenotypes, investigate possible genetic compensation through transcriptomic or proteomic comparisons with wildtype strains. Complementation studies using controlled expression systems can help establish dose-dependent effects.

What are the current knowledge gaps in SAR0874 research?

Despite progress in characterizing SAR0874, significant knowledge gaps remain in understanding this protein's precise biological function. The exact biochemical mechanism of SAR0874 action remains undefined, with limited information about potential enzymatic activities or specific binding partners. The three-dimensional structure has not been experimentally determined, limiting structure-based functional predictions. The regulatory network controlling SAR0874 expression under different conditions is incompletely characterized, as are the specific stress signals that modulate its activity .

Research is needed to clarify how SAR0874 interacts with other cellular components and pathways, particularly in relation to stress responses and potential virulence mechanisms. The contribution of SAR0874 to host-pathogen interactions remains largely unexplored, with limited data on its role during infection processes. Future studies should address these gaps through integrated approaches combining structural biology, systems-level analyses, and in vivo infection models. Collaborative research combining expertise in protein biochemistry, microbial physiology, and infection biology will be essential to fully elucidate the biological significance of this conserved staphylococcal protein.

How might future research applications of SAR0874 develop?

Future research applications involving SAR0874 may extend in several promising directions as our understanding of this protein deepens. If SAR0874 is confirmed to play significant roles in stress responses or virulence, it could become a target for novel antimicrobial development, particularly if structural studies reveal druggable sites. The protein might serve as a biomarker for specific physiological states in S. aureus, potentially useful for diagnostic applications or monitoring treatment responses .