Recombinant Staphylococcus aureus UPF0382 membrane protein SAR0588 (SAR0588)

What is Recombinant Staphylococcus aureus UPF0382 Membrane Protein SAR0588?

Recombinant Staphylococcus aureus UPF0382 membrane protein SAR0588 is a bacterial membrane protein originally identified in Staphylococcus aureus strain MRSA252, with UniProt accession number Q6GJ86 . This protein belongs to the UPF0382 family of uncharacterized membrane proteins and consists of 122 amino acids forming a transmembrane structure. The recombinant form is produced through molecular cloning techniques, where the SAR0588 gene is isolated from S. aureus, inserted into an expression vector, and expressed in a suitable host system to obtain purified protein for research purposes. The protein plays a potential role in the membrane structure and function of S. aureus, which is a significant human pathogen responsible for various infections ranging from minor skin infections to life-threatening conditions such as bacteremia and endocarditis .

What are the structural characteristics and properties of SAR0588 protein?

SAR0588 is a 122-amino acid transmembrane protein with a predominantly hydrophobic composition, consistent with its membrane-associated nature . The amino acid sequence (MKLFIILGALNAMMAVGTGAFGAHGLQGKISDHYLSVWEKATTYQMYHGLALLIIGVISGTTSINVNWAGWLIFAGIIFFSGSLYILVLTQIKVLGAITPIGGVLFIIGWIMLIIATFKFAG) reveals multiple hydrophobic regions interspersed with charged residues that likely contribute to membrane anchoring and protein function .

Analysis of the protein sequence suggests the following structural properties:

The recombinant form is typically produced with affinity tags to facilitate purification, with the tag type determined during the production process . The protein is most stable when stored in a Tris-based buffer with 50% glycerol at -20°C or -80°C for extended storage, with working aliquots maintained at 4°C for up to one week .

What are the recommended storage and handling conditions for recombinant SAR0588?

For optimal stability and activity of recombinant SAR0588 protein, the following storage and handling procedures should be implemented:

• Storage buffer composition: Use Tris-based buffer containing 50% glycerol which has been optimized specifically for this membrane protein .

• Long-term storage: Store at -20°C, or preferably at -80°C for extended periods to minimize protein degradation .

• Working conditions: Maintain working aliquots at 4°C for up to one week to reduce freeze-thaw cycles .

• Freeze-thaw considerations: Repeated freezing and thawing should be avoided as this can lead to protein denaturation and loss of structural integrity. It is recommended to prepare small working aliquots during initial thawing .

• Handling precautions: As a membrane protein, SAR0588 has hydrophobic regions that can promote aggregation when exposed to aqueous environments. Consider adding mild detergents or stabilizing agents when working with the protein in solution.

• Quality control: Before experimental use, verify protein integrity through methods such as SDS-PAGE, Western blotting, or mass spectrometry to ensure the full-length protein (122 amino acids) is present and intact.

What expression systems are most effective for producing recombinant SAR0588?

The expression of membrane proteins like SAR0588 presents significant challenges due to their hydrophobic nature and potential toxicity to host cells. Based on established protocols for similar membrane proteins, researchers should consider the following expression systems and methodologies:

• Prokaryotic expression systems:

  • While E. coli is commonly used for protein expression, membrane proteins often encounter expression challenges including protein hydrophobicity, codon rarity, and potential toxicity to the host cell .

  • For SAR0588 expression in E. coli, consider using specialized strains such as C41(DE3) or C43(DE3) that are adapted for membrane protein expression.

  • Codon optimization of the SAR0588 gene sequence for E. coli usage can significantly improve expression levels, especially considering the different codon usage between S. aureus and E. coli .

• Eukaryotic expression systems:

  • For more complex structural studies, consider yeast systems (Pichia pastoris or Saccharomyces cerevisiae) which provide a eukaryotic membrane environment.

  • Insect cell expression using baculovirus systems may offer advantages for folding and post-translational modifications.

• Cell-free expression systems:

  • These systems can be particularly advantageous for membrane proteins as they circumvent toxicity issues and allow direct incorporation into liposomes or nanodiscs.

• Expression optimization strategies:

  Strategy	Method	Rationale
  Fusion tags	Use solubility-enhancing fusion partners (MBP, SUMO)	Improves solubility and facilitates detection
  Temperature control	Express at lower temperatures (16-25°C)	Slows protein synthesis and improves folding
  Inducer concentration	Optimize IPTG or other inducer levels	Controls expression rate to prevent aggregation
  Media supplements	Add glycerol, osmolytes, or specific lipids	Stabilizes membrane proteins during expression
  Dual-tag approach	N and C-terminal tags	Enables selection of full-length protein

• Verification of full-length expression:

  • Use expression vectors with fusion tags on both the N and C-termini to distinguish full-length proteins from truncated products .

  • Increase imidazole concentration during elution to ensure selection of complete proteins if using His-tag purification .

What purification strategies are most effective for obtaining high-quality SAR0588 protein?

Purifying membrane proteins presents distinct challenges compared to soluble proteins. For SAR0588, a methodical approach incorporating the following strategies is recommended:

• Membrane extraction:

  • Begin with efficient cell lysis using methods that preserve membrane integrity (sonication or French press).

  • Extract membrane fractions through differential centrifugation.

  • Solubilize the membrane fraction using appropriate detergents selected based on the downstream application.

• Detergent selection:

  Detergent Class	Examples	Advantages	Considerations
  Mild non-ionic	DDM, LMNG	Gentle, maintains protein structure	May have lower extraction efficiency
  Zwitterionic	CHAPS, Fos-choline	Effective solubilization	Can be more denaturing
  Steroid-based	Digitonin, CHS	Preserves protein-protein interactions	More expensive, less efficient

• Affinity chromatography:

  • Utilize the tag incorporated during expression (His, GST, MBP) for initial capture .

  • Include low concentrations of detergent in all buffers to maintain protein solubility.

  • Consider using on-column detergent exchange if the downstream application requires a different detergent environment.

• Secondary purification:

  • Size exclusion chromatography (SEC) to separate monomeric protein from aggregates and to assess protein homogeneity.

  • Ion exchange chromatography as an additional purification step if higher purity is required.

• Alternative approaches for structural and functional studies:

  • Consider reconstitution into nanodiscs or proteoliposomes for studies requiring a lipid bilayer environment.

  • The MNP (membrane nanoparticle) platform can be employed to extract high-purity nanoscale cell membrane particles while maintaining the conformation and activity of membrane proteins like SAR0588 .

• Quality assessment:

  • Circular dichroism to verify secondary structure.

  • Thermal stability assays to optimize buffer conditions.

  • Dynamic light scattering to assess homogeneity and aggregation status.

  • Mass spectrometry to confirm protein identity and integrity.

How can researchers effectively study SAR0588 structure-function relationships?

Understanding the structure-function relationship of SAR0588 requires a multidisciplinary approach combining computational predictions with experimental validation:

• Computational structure prediction:

  • Utilize AI-based protein structure prediction tools like AlphaFold2 to generate initial structural models of SAR0588 .

  • Perform molecular dynamics simulations to understand membrane integration and dynamics.

  • Identify conserved residues through multiple sequence alignment with UPF0382 family proteins from other bacterial species.

• Experimental structure determination:

  • X-ray crystallography: Requires generating crystal-grade protein, which is challenging for membrane proteins but provides high-resolution structures.

  • Cryo-electron microscopy: Increasingly popular for membrane proteins, especially when reconstituted in nanodiscs.

  • NMR spectroscopy: Useful for smaller membrane proteins or domains, providing dynamics information.

• Functional analysis methodologies:

  Technique	Application	Information Gained
  Site-directed mutagenesis	Alter specific residues	Identify functionally important amino acids
  Fluorescence-based assays	Monitor conformational changes	Detect ligand binding or protein interactions
  Electrophysiology	Study ion transport	Determine if SAR0588 has channel or transporter activity
  FRET/BRET	Protein-protein interactions	Identify binding partners in the membrane
  Isothermal titration calorimetry	Binding studies	Quantify interactions with potential ligands

• In vivo functional studies:

  • Generate SAR0588 knockout strains in S. aureus to assess phenotypic changes.

  • Complement with wild-type and mutant versions to validate functional hypotheses.

  • Assess impact on virulence in infection models when the protein is absent or altered.

• Integration with systems biology approaches:

  • Combine with transcriptomics or proteomics data to place SAR0588 in broader cellular pathways.

  • Network analysis to predict functional associations based on co-expression patterns.

What role might SAR0588 play in S. aureus pathogenesis and virulence?

While the specific function of SAR0588 has not been fully characterized in the provided search results, its nature as a membrane protein in a significant human pathogen suggests several potential roles in S. aureus pathogenesis:

• Membrane integrity and cellular physiology:

  • As a membrane protein, SAR0588 likely contributes to membrane structure, stability, or function in S. aureus.

  • It may play a role in adapting to environmental changes encountered during infection, such as pH, temperature, or osmotic stress.

• Potential role in virulence:

  • Many S. aureus membrane proteins are involved in virulence mechanisms, including adhesion to host tissues, immune evasion, or nutrient acquisition .

  • SAR0588 might contribute to one or more of the following virulence processes:

    • Biofilm formation, which enhances antibiotic resistance and immune evasion

    • Adaptation to the host environment during infection

    • Transport of virulence factors or nutrients essential for pathogenesis

• Antibiotic resistance considerations:

  • Membrane proteins can contribute to antibiotic resistance by altering membrane permeability or participating in efflux systems .

  • SAR0588 is found in MRSA252, a methicillin-resistant strain, suggesting potential involvement in the resistance phenotype .

• Immune interaction potential:

  • S. aureus employs various mechanisms to evade or manipulate host immune responses .

  • As a surface-exposed protein, SAR0588 might interact with host immune components, potentially contributing to the bacterium's ability to persist during infection.

• Comparative analysis with other pathogens:

  • Investigation of UPF0382 family proteins in other bacterial species could provide insights into conserved functions relevant to bacterial survival or pathogenesis.

How might SAR0588 be exploited for vaccine development or antimicrobial strategies?

The persistent challenges in developing effective vaccines against S. aureus highlight the need for novel approaches. SAR0588, as a membrane protein, represents a potential target for both vaccine development and antimicrobial strategies:

• Vaccine development considerations:

  • No vaccine for S. aureus has been approved despite extensive research efforts , indicating the need for innovative approaches.

  • Membrane proteins like SAR0588 can serve as vaccine antigens if they are:

    • Conserved across different S. aureus strains

    • Surface-exposed and accessible to antibodies

    • Expressed during infection

    • Involved in virulence or survival mechanisms

• Multi-antigen vaccine approaches:

  • Successful S. aureus vaccine strategies often require multiple antigens targeting different virulence mechanisms .

  • SAR0588 could potentially be included in multi-component vaccines similar to the recombinant five-antigen S. aureus vaccine (rFSAV) which incorporated multiple virulence factors .

  Current rFSAV Components	Function	Potential Complementarity with SAR0588
  Staphylococcal protein A (SpA)	Immune evasion	Could complement with membrane integrity functions
  α-hemolysin (Hla)	Cytotoxin	Targets different aspects of pathogenesis
  Iron surface determinant B (IsdB-N2)	Iron acquisition	Addresses different survival mechanisms
  Staphylococcal enterotoxin B (SEB)	Superantigen	Different immunological targets
  Manganese transport protein C (MntC)	Metal acquisition	Different metabolic pathways

• Antimicrobial development strategies:

  • If SAR0588 proves essential for S. aureus survival or virulence, it could be targeted for antimicrobial development.

  • Potential approaches include:

    • Small molecule inhibitors disrupting protein function

    • Peptide mimetics interfering with protein-protein interactions

    • Antibody-antibiotic conjugates targeting the protein while delivering antimicrobial agents

• Drug delivery applications:

  • Liposomes or nanoparticles decorated with SAR0588-targeting ligands could enhance delivery of antimicrobials specifically to S. aureus.

  • This approach might improve efficacy while reducing off-target effects of antimicrobial therapy.

• Immunomodulatory strategies:

  • Understanding SAR0588 interactions with host immunity could inform development of immunomodulatory therapies that enhance natural clearance of S. aureus infections.

What analytical techniques are most informative for studying SAR0588 interactions with host components?

Understanding how SAR0588 interacts with host components requires sophisticated analytical approaches that preserve protein structure while enabling detection of specific interactions:

• In vitro binding studies:

  • Surface plasmon resonance (SPR) to quantify binding kinetics with purified host components.

  • Enzyme-linked immunosorbent assays (ELISA) using recombinant SAR0588 to screen for potential interactions with host factors .

  • Pull-down assays with tagged SAR0588 to identify binding partners from host cell lysates.

• Cellular interaction studies:

  Technique	Application	Insights Provided
  Flow cytometry	Cell binding	Quantification of SAR0588 binding to different host cell types
  Confocal microscopy	Localization	Visualization of where SAR0588 localizes on host cells
  FRET analysis	Protein proximity	Detection of close interactions with specific host proteins
  Cell-based reporter assays	Signaling	Identification of host signaling pathways activated by SAR0588

• Structural biology approaches:

  • Co-crystallization of SAR0588 with identified binding partners.

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces.

  • Cryo-EM analysis of SAR0588 in complex with larger host components.

• Systems biology integration:

  • Transcriptomic analysis of host cells exposed to purified SAR0588.

  • Proteomics to identify changes in host protein expression or post-translational modifications.

  • Pathway analysis to contextualize identified interactions within host response networks.

• Animal model validation:

  • Comparison of wild-type S. aureus versus SAR0588 knockout strains in infection models.

  • Immunological profiling to characterize host response differences.

  • Tissue-specific analyses to identify critical sites of SAR0588-host interactions during infection.

How might high-throughput screening approaches be applied to SAR0588 research?

High-throughput screening (HTS) methodologies can accelerate discovery of SAR0588 functions, interactions, and potential inhibitors:

• Functional screening approaches:

  • Phenotypic screening of SAR0588 mutant libraries to identify functionally important residues.

  • Growth condition arrays to determine environmental factors influencing SAR0588 expression or function.

  • Bacterial two-hybrid systems to identify prokaryotic protein interaction partners.

• Compound screening strategies:

  • Fragment-based screening to identify chemical scaffolds that bind to SAR0588.

  • Virtual screening using computational models of SAR0588 structure to identify potential binding pockets and ligands.

  • Repurposing screens with approved drug libraries to identify compounds that might interact with SAR0588.

• Advanced HTS methodologies:

  Approach	Implementation	Expected Outcomes
  Microfluidic systems	Droplet-based assays	Rapid testing of multiple conditions with minimal protein consumption
  Surface display technologies	Phage or yeast display of SAR0588 variants	Identification of variants with altered binding properties
  Deep mutational scanning	Comprehensive mutant libraries	Mapping of sequence-function relationships across the entire protein
  Label-free binding detection	Mass spectrometry, interferometry	Direct detection of interactions without requiring labeled components

• Biosensor development:

  • Engineer SAR0588-based biosensors by incorporating fluorescent reporters or electrochemical elements.

  • These could be used both for HTS and for developing diagnostic applications for S. aureus detection.

• Integration with artificial intelligence:

  • Machine learning approaches to predict SAR0588 interactions based on sequence features.

  • AI-assisted design of selective inhibitors targeting unique features of SAR0588 structure .

How does SAR0588 compare to membrane proteins in other Staphylococcal species?

Comparative analysis of SAR0588 across Staphylococcal species provides evolutionary context and may reveal important functional insights:

• Evolutionary conservation analysis:

  • Sequence alignment of SAR0588 homologs across Staphylococcal species to identify conserved and variable regions.

  • Phylogenetic analysis to understand the evolutionary history of this protein family.

  • Assessment of selection pressure on different protein domains to identify functionally critical regions.

• Structural comparison methodologies:

  • Homology modeling of SAR0588 variants from different species.

  • Molecular dynamics simulations to compare structural stability and dynamics.

  • Identification of species-specific structural features that might relate to host adaptation.

• Functional comparison approaches:

  • Cross-species complementation studies to determine functional conservation.

  • Comparative expression analysis under different environmental conditions.

  • Host interaction studies with SAR0588 variants from different Staphylococcal species with varying host ranges.

• Pathogenesis-related comparisons:

  Species	Pathogenicity	SAR0588 Homolog Characteristics	Potential Functional Implications
  S. aureus	Major human pathogen	Original protein of interest	Associated with human infections
  S. epidermidis	Opportunistic pathogen	Likely highly similar	May contribute to skin colonization
  S. lugdunensis	Emerging pathogen	Variant may have unique features	Could relate to virulence differences
  S. saprophyticus	Urinary tract infections	Potential specialization	May reflect urinary tract adaptation
  Coagulase-negative staphylococci	Generally less virulent	Possible structural differences	Could explain virulence variation

• Genome context analysis:

  • Examination of the genomic neighborhood of SAR0588 homologs across species.

  • Identification of co-evolved genes that might functionally interact with SAR0588.

  • Analysis of regulatory elements to understand expression control mechanisms.

What are the most promising applications of SAR0588 research in addressing S. aureus infections?

The study of SAR0588 offers several promising avenues for addressing the significant clinical challenges posed by Staphylococcus aureus infections:

• Diagnostic applications:

  • Development of SAR0588-based detection methods for rapid identification of S. aureus, particularly MRSA strains.

  • Generation of specific antibodies against SAR0588 for immunodiagnostic applications.

  • Potential inclusion in multi-target diagnostic panels for improved sensitivity and specificity.

• Therapeutic targeting:

  • If SAR0588 proves essential for bacterial survival or virulence, it represents a novel target for antimicrobial development.

  • The membrane location makes it potentially accessible to antibody-based therapies or targeted drug delivery systems.

  • Structure-based drug design approaches could yield selective inhibitors with reduced likelihood of cross-resistance to existing antibiotics.

• Vaccine development:

  • Inclusion in multi-component vaccine strategies, potentially complementing the approaches used in the recombinant five-antigen S. aureus vaccine .

  • If conserved across strains, SAR0588 could contribute to broad protection against diverse S. aureus isolates.

  • Understanding of host immune responses to SAR0588 could inform adjuvant selection and delivery approaches.

• Fundamental understanding of S. aureus pathogenesis:

  • Elucidation of SAR0588 function may reveal novel aspects of S. aureus membrane biology and adaptation to host environments.

  • This knowledge could inform broader antimicrobial strategies targeting membrane integrity or function.

  • Insights from SAR0588 studies may be applicable to other bacterial pathogens with similar membrane proteins.

• Addressing antibiotic resistance:

  • With the significant challenge of antimicrobial resistance in S. aureus, including MRSA , novel targets like SAR0588 represent opportunities for developing antibiotics with new mechanisms of action.

  • Combination approaches targeting SAR0588 alongside other bacterial components might reduce the development of resistance.