Recombinant Staphylococcus aureus Uncharacterized protein SAS0717 (SAS0717)

What Expression Systems Are Most Effective for Producing Recombinant SAS0717?

For optimal expression of SAS0717, E. coli-based systems have demonstrated considerable success with similar S. aureus membrane proteins. Based on protocols used for comparable uncharacterized membrane proteins (such as SAS0711), we recommend:

Recommended Expression Protocol:

• Expression vector: pET32a or pET28a with T7 promoter

• Host strain: BL21(DE3) or Rosetta(DE3)

• Induction: 0.5-1.0 mM IPTG

• Temperature: 18-25°C post-induction (to minimize inclusion body formation)

• Duration: 12-16 hours

Research with similar S. aureus proteins shows that inclusion body formation is common with membrane proteins, necessitating optimization of solubilization conditions. A step-gradient purification approach using histidine tags has proven effective for similar uncharacterized S. aureus proteins .

How Should I Approach Initial Structural Characterization of SAS0717?

Begin with bioinformatic analysis followed by empirical structural studies:

Methodological Workflow:

• Bioinformatic prediction:

  • Transmembrane domain prediction using TMHMM or Phobius

  • Secondary structure prediction via PSIPRED

  • Homology modeling using I-TASSER or Swiss-Model

• Experimental verification:

  • Circular dichroism (CD) spectroscopy for secondary structure determination

  • Limited proteolysis to identify domain boundaries

  • X-ray crystallography or cryo-EM for tertiary structure (if protein quantity and quality permit)

Similar uncharacterized S. aureus membrane proteins frequently contain multiple transmembrane helices. For instance, SAS0711 contains motifs characteristic of transport proteins with distinct N- and C-terminal domains .

What Are Effective Methods for Determining SAS0717 Subcellular Localization?

Confirming subcellular localization requires multiple complementary approaches:

Recommended Protocol Combination:

For S. aureus surface proteins, researchers have successfully employed anti-His-DUF1542 antisera in immunofluorescence microscopy to visualize surface localization, as demonstrated with SasC protein .

What Functional Assays Should I Consider to Determine the Role of SAS0717?

The function of uncharacterized S. aureus proteins can be investigated through systematic phenotypic analyses:

Functional Characterization Strategy:

• Cell aggregation and biofilm formation assays:

  • Crystal violet staining of adherent biofilms (A₄₉₀ quantification)

  • Cell aggregation visualization in liquid culture

  • Flow cell chamber biofilm formation with confocal microscopy

• Host-interaction studies:

  • Binding to extracellular matrix proteins (fibrinogen, fibronectin, collagen)

  • Platelet aggregation assays

  • Neutrophil interaction studies

• Virulence assessment:

  • Murine infection models comparing wild-type and knockout strains

  • Cytotoxicity assays with relevant host cell types

  • IL-1β production in macrophages

Research with other S. aureus surface proteins has revealed their importance in intercellular adhesion and biofilm formation. For example, SasC's N-terminal domain was found to confer cell aggregation and enhanced biofilm formation, while knockout mutants showed reduced biofilm formation capabilities .

How Can I Determine If SAS0717 Is Involved in Immune Evasion?

Many S. aureus proteins contribute to immune evasion strategies. To investigate this possibility for SAS0717:

Immune Evasion Assessment Protocol:

• Neutrophil interaction studies:

  • Neutrophil chemotaxis assays in presence/absence of recombinant protein

  • Phagocytosis inhibition assays

  • Neutrophil extracellular trap (NET) formation assessment

• Complement interaction analysis:

  • Complement activation assays (classical, alternative pathways)

  • Complement deposition on bacterial surface

  • Direct binding to complement components

• Receptor binding studies:

  • Flow cytometry to detect binding to immune cell receptors

  • Surface plasmon resonance to measure binding kinetics

  • Competitive binding assays with known ligands

Recent research has identified previously uncharacterized S. aureus proteins like SSL6 and SElX that bind to neutrophil surface receptors. SSL6 was found to bind CD47, while SElX binds PSGL-1, inhibiting neutrophil recruitment . Similar approaches could reveal immune modulatory functions of SAS0717.

What Genomic Approaches Can I Use to Study the Expression Profile of SAS0717?

Understanding when and where SAS0717 is expressed provides crucial contextual information:

Expression Analysis Framework:

• Transcriptional profiling:

  • qRT-PCR during different growth phases

  • RNA-Seq under various environmental conditions

  • Promoter analysis using reporter fusions

• Regulon analysis:

  • ChIP-seq to identify transcription factor binding

  • Differential expression following regulatory gene deletion

  • Identification of co-regulated genes

• In vivo expression:

  • RNA extraction from infected tissues

  • In vivo expression technology (IVET)

  • Animal infection models with reporter strains

S. aureus virulence factors often show growth phase-dependent expression. For example, SasC production has been detected throughout growth in S. aureus strains, with variable expression levels at different growth phases .

How Can I Generate and Validate a SAS0717 Knockout Mutant?

Creating a clean knockout mutant is essential for definitive functional studies:

Knockout Generation Protocol:

• Allelic replacement strategy:

  • Design constructs with 500-1000bp homology arms flanking SAS0717

  • Include selectable marker (e.g., antibiotic resistance)

  • Transform into S. aureus using electroporation

  • Screen transformants for double crossover events

• Validation approaches:

  • PCR verification of gene deletion

  • RT-PCR confirmation of transcript absence

  • Western blot to confirm protein absence

  • Complementation with wild-type gene to restore phenotype

• Phenotypic characterization:

  • Growth curve analysis under various conditions

  • Biofilm formation capacity

  • Virulence in animal models

Studies with SasC mutants demonstrated reduced biofilm formation compared to wild-type strains under various conditions, including different carbohydrate supplementation. The SasC Tn917 insertion mutant (SMH2035) showed consistently reduced biofilm formation with A₄₉₀ values decreasing from 1.8 to 1.6 with glucose addition and from 1.0 to 0.5 without carbohydrate supplementation .

What Approaches Can Determine If SAS0717 Is a Potential Vaccine Target?

Evaluating vaccine potential requires a systematic assessment of immunogenicity and protective capacity:

Vaccine Potential Assessment Strategy:

• Immunogenicity studies:

  • Recombinant protein immunization in mice

  • Antibody titer determination

  • Epitope mapping

• Protection assays:

  • Challenge studies with virulent S. aureus in immunized animals

  • Assessment of bacterial load in tissues

  • Measurement of inflammatory markers

• Cross-protection evaluation:

  • Testing against multiple S. aureus strains

  • Determination of conservation across clinical isolates

  • Evaluation of synergy with other antigen candidates

Fusion proteins combining multiple S. aureus antigens have shown promising results. For example, chimeric proteins combining FnBP+ClfA (FC) induced high antibody levels in mice and provided protection against S. aureus challenge, with significantly reduced bacterial loads in spleen and liver tissues .

How Can I Investigate Protein-Protein Interactions Involving SAS0717?

Identifying interaction partners is crucial for understanding protein function:

Interaction Analysis Methods:

• Pull-down assays:

  • His-tag or GST-tag affinity purification

  • Co-immunoprecipitation with specific antibodies

  • Cross-linking followed by mass spectrometry

• Two-hybrid systems:

  • Bacterial two-hybrid for prokaryotic interactions

  • Yeast two-hybrid for eukaryotic host interactions

  • Split-GFP complementation assays

• Advanced biophysical methods:

  • Surface plasmon resonance (SPR)

  • Isothermal titration calorimetry (ITC)

  • Microscale thermophoresis (MST)

Studies with S. aureus protein A demonstrated its ability to bind to the Fc portion of immunoglobulins from various species. Similar interaction studies could reveal binding partners for SAS0717, potentially identifying roles in host-pathogen interactions .

What Bioinformatic Tools Should I Use to Predict Functional Domains in SAS0717?

Computational analysis can provide valuable insights into potential functions:

Bioinformatic Analysis Pipeline:

• Sequence-based analysis:

  • BLAST against characterized proteins

  • Conserved domain search (CDD, Pfam)

  • Motif identification using MEME, GLAM2

• Structural predictions:

  • AlphaFold or RoseTTAFold for 3D structure prediction

  • Prediction of binding pockets and active sites

  • Molecular dynamics simulations to assess flexibility

• Evolutionary analysis:

  • Phylogenetic profiling

  • Identification of co-evolving residues

  • Positive selection analysis

Uncharacterized S. aureus membrane proteins often contain conserved domains with functional implications. For example, SasC contains a repeat region with 17 repeats of 72 amino acids similar to the DUF1542 domain, as well as a FIVAR motif in its N-terminal region that was found to mediate cell aggregation and biofilm formation .

How Should I Design Experiments to Study SAS0717's Role in S. aureus Pathogenesis?

A comprehensive approach combining in vitro and in vivo studies provides the most robust insights:

Pathogenesis Investigation Framework:

• In vitro infection models:

  • Epithelial cell adhesion and invasion assays

  • Macrophage survival and cytokine production

  • Biofilm formation on relevant substrates

• Ex vivo tissue models:

  • Human skin explant infection

  • Blood survival assays

  • Whole blood killing assays

• In vivo infection models:

  • Murine bacteremia and organ abscess models

  • Skin infection models

  • Influenza superinfection models

Recent research has shown that certain S. aureus surface proteins play crucial roles in specific infection contexts. For example, SasD was found to induce changes in both inflammatory and homeostatic lung markers during pneumonia and influenza superinfection, with SasD mutants demonstrating decreased bacterial burden, inflammatory responses, and mortality compared to wild-type S. aureus .

What Techniques Can Identify Post-Translational Modifications of SAS0717?

Post-translational modifications can significantly impact protein function:

PTM Analysis Methods:

For S. aureus surface proteins, it's particularly important to analyze potential signal peptide cleavage and cell wall anchoring mechanisms. Analysis of similar proteins has identified LPXTG motifs that serve as recognition sites for sortase enzymes, facilitating covalent attachment to peptidoglycan .