Recombinant Staphylococcus aureus UPF0060 membrane protein SAB2216c (SAB2216c)

What is SAB2216c protein and what are its structural characteristics?

SAB2216c is a membrane protein from Staphylococcus aureus classified as part of the UPF0060 protein family. It consists of 108 amino acids with a complete sequence of MLYPIFIFILAGLCEIGGGYLIWLWLREGQCSLVGLIGGAILMLYGVIATFQSFPSFGRVYAAYGGVFIIMSLIFAMVVDKQMPDKYDVIGAIICIVGVLVMLLPSRA . As a membrane protein, SAB2216c exhibits hydrophobic regions that facilitate its integration into cellular membranes. The protein's structure suggests it contains multiple transmembrane domains, which is characteristic of integral membrane proteins that span the lipid bilayer.

How is recombinant SAB2216c protein typically produced for research purposes?

Recombinant SAB2216c protein is commonly produced using E. coli expression systems with an N-terminal His-tag for purification purposes . The full-length protein (1-108 amino acids) is expressed in E. coli, then purified using affinity chromatography techniques that leverage the His-tag. The purified protein is typically provided as a lyophilized powder with greater than 90% purity as determined by SDS-PAGE . For research applications, the protein is reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, often with added glycerol (5-50% final concentration) for long-term storage stability .

What are the optimal storage conditions for recombinant SAB2216c protein?

For optimal stability, recombinant SAB2216c protein should be stored at -20°C to -80°C upon receipt, with aliquoting necessary for multiple use scenarios to avoid repeated freeze-thaw cycles . Working aliquots can be stored at 4°C for up to one week . The protein is typically provided in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0 , or alternatively in a Tris-based buffer with 50% glycerol optimized for protein stability . These storage conditions are designed to maintain protein integrity and activity for experimental applications.

What experimental applications is recombinant SAB2216c protein suitable for?

Recombinant SAB2216c protein is suitable for a range of biochemical and structural biology applications including:

• Protein-protein interaction studies

• Antibody generation and validation

• Structural characterization of membrane proteins

• Functional assays to determine biological activity

• ELISA-based detection and quantification methods

The His-tagged format facilitates detection and purification in various experimental setups, making it versatile for different research protocols.

What experimental design considerations should be made when studying SAB2216c membrane insertion mechanisms?

When designing experiments to study SAB2216c membrane insertion mechanisms, researchers should consider several critical factors:

• Membrane mimetic systems selection: Choose between liposomes, nanodiscs, or detergent micelles based on experimental goals. Liposomes offer a native-like environment but may present challenges for structural studies, while nanodiscs provide a more controlled system that's compatible with various biophysical techniques.

• Protein labeling strategies: Consider site-specific fluorescent or spin labeling at non-conserved residues to minimize functional disruption. Based on the amino acid sequence (MLYPIFIFILAGLCEIGGGYLIWLWLREGQCSLVGLIGGAILMLYGVIATFQSFPSFGRVYAAYGGVFIIMSLIFAMVVDKQMPDKYDVIGAIICIVGVLVMLLPSRA), cysteine residues can be introduced for specific labeling approaches .

• Controls and variables: Implement proper experimental controls including:

  • Non-membrane inserting protein controls

  • Lipid composition variation (3+ compositions)

  • pH and ionic strength conditions (minimum of 3 conditions)

  • Temperature variation effects

For quantitative assessment, fluorescence spectroscopy, circular dichroism, or FRET-based approaches can track insertion kinetics and thermodynamics, requiring at least three independent experimental replicates for statistical validation.

How can researchers address the challenges of SAB2216c protein aggregation during structural studies?

SAB2216c protein aggregation presents a significant challenge for structural biology studies due to its highly hydrophobic nature. To mitigate this issue:

• Buffer optimization strategy:

  • Systematically screen buffers with varying pH (6.0-8.5), salt concentrations (100-500 mM NaCl), and additives

  • Include mild solubilizing agents such as glycerol (5-20%)

  • Test the addition of non-ionic detergents (0.01-0.1%)

  • Consider adding stabilizing agents like trehalose (3-10%)

• Protein engineering approach:

  • Design fusion constructs with solubility-enhancing partners

  • Identify and mutate aggregation-prone regions based on sequence analysis

  • Consider truncation constructs that maintain functional domains

• Sample preparation protocol:

  • Maintain low protein concentrations during initial refolding steps

  • Employ step-wise dialysis to gradually remove denaturants

  • Use analytical size exclusion chromatography to monitor aggregation states

  • Implement temperature control during all handling steps

Implementing these strategies can significantly reduce aggregation while maintaining native-like protein structure, crucial for obtaining reliable structural data.

What are the appropriate experimental controls when investigating SAB2216c interactions with other Staphylococcus aureus proteins?

When investigating protein-protein interactions involving SAB2216c, implementing rigorous controls is essential for data validity:

• Positive and negative interaction controls:

  • Positive control: Use a well-characterized membrane protein interaction pair

  • Negative control: Include an unrelated membrane protein from S. aureus

  • Tag-only control: Express and purify the tag portion alone to rule out tag-mediated interactions

• Methodological validation controls:

  Control Type	Purpose	Implementation
  Method specificity	Validate assay performance	Include known interacting proteins
  Buffer composition	Rule out buffer-induced artifacts	Test multiple buffer conditions
  Concentration dependence	Assess binding kinetics	Perform serial dilutions (minimum 5 concentrations)
  Binding site mutation	Confirm specific binding	Mutate predicted interaction interfaces
  Competitive binding	Verify binding specificity	Add unlabeled protein as competitor

• Data analysis approach:

  • Apply multiple complementary interaction methods (pull-down, SPR, crosslinking)

  • Quantify binding using at least three biological replicates

  • Plot binding curves with appropriate statistical analysis

  • Validate key interactions using cellular co-localization studies

These controls ensure that observed interactions are specific to SAB2216c rather than experimental artifacts, significantly increasing confidence in research findings.

How can researchers design experiments to elucidate the potential role of SAB2216c in Staphylococcus aureus virulence?

To investigate SAB2216c's potential role in S. aureus virulence, a systematic experimental approach should be designed:

• Gene knockout and complementation studies:

  • Generate SAB2216c deletion mutants in relevant S. aureus strains

  • Create complemented strains expressing wild-type SAB2216c

  • Develop point mutations in conserved domains for structure-function analysis

  • Assess growth curves under various stress conditions (minimum 3 replicates)

• Phenotypic characterization protocol:

  • Evaluate membrane integrity through permeability assays

  • Assess biofilm formation capability using crystal violet staining

  • Measure susceptibility to antimicrobial peptides at multiple concentrations

  • Quantify adherence to host cell lines (minimum 3 cell types)

  • Monitor survival within macrophages at multiple time points (2, 4, 8, 24 hours)

• In vivo infection models:

  • Compare wild-type and mutant strains in multiple infection models

  • Measure bacterial burden in tissues at defined time points

  • Assess host immune response through cytokine profiling

  • Evaluate histopathological changes in infected tissues

This comprehensive approach combines molecular, cellular, and in vivo methods to systematically determine SAB2216c's contribution to S. aureus pathogenesis, addressing potential functional redundancy that often complicates virulence factor studies.

What experimental design is recommended for determining if SAB2216c forms functional complexes with other membrane proteins?

To investigate SAB2216c's potential to form functional complexes with other membrane proteins, a multi-disciplinary experimental design is recommended:

• Co-purification and native complex isolation:

  • Implement mild solubilization conditions using digitonin or LMNG detergents

  • Perform blue native PAGE to identify potential complexes

  • Use size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to determine complex stoichiometry

  • Validate interactions through co-immunoprecipitation with specific antibodies

• Cross-linking coupled mass spectrometry approach:

  • Apply membrane-permeable crosslinkers at optimized concentrations

  • Digest cross-linked complexes with multiple proteases for better coverage

  • Analyze by LC-MS/MS with specialized cross-link identification software

  • Map interaction interfaces using the known SAB2216c sequence

• Functional reconstitution experiments:

  Approach	Measurements	Controls Required
  Proteoliposome reconstitution	Transport/channel activity	Protein-free liposomes
  Electrical measurements	Conductance changes	Single protein controls
  FRET-based proximity	Energy transfer efficiency	Non-interacting protein pairs
  Cryo-EM structural analysis	Complex architecture	Individual protein samples

• Validation in native membranes:

  • Perform super-resolution microscopy to track co-localization

  • Implement FRAP (Fluorescence Recovery After Photobleaching) to assess mobility

  • Use APEX2 proximity labeling to identify neighbors in native environment

This comprehensive strategy combines biochemical, biophysical, and imaging approaches to establish both the existence and functional significance of SAB2216c-containing membrane protein complexes.

What are the recommended protocols for optimizing recombinant SAB2216c expression and purification?

For researchers seeking to optimize SAB2216c expression and purification, a systematic approach addressing the protein's membrane-associated nature is essential:

• Expression system optimization:

  • Compare expression levels in multiple E. coli strains (BL21(DE3), C41(DE3), Rosetta)

  • Test induction conditions (IPTG concentration: 0.1-1.0 mM; temperature: 16°C, 25°C, 37°C)

  • Evaluate different culture media (LB, TB, auto-induction media)

  • Optimize expression time (4h, 8h, overnight)

• Membrane protein extraction strategy:

  • Screen detergents for solubilization (DDM, LMNG, digitonin)

  • Test detergent concentrations (1-5× CMC)

  • Optimize solubilization time and temperature

  • Include protease inhibitors to prevent degradation

• Purification protocol refinement:

  • Implement two-step purification (IMAC followed by size exclusion)

  • Test different imidazole concentration gradients for elution

  • Evaluate buffer compositions for stability (pH 7.0-8.5)

  • Consider on-column detergent exchange if necessary

The purified protein should achieve >90% purity as verified by SDS-PAGE , with typical yields of 2-5 mg per liter of culture. Store the final product as described in section 1.3, with recommended reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

How should researchers design experiments to investigate SAB2216c topology and membrane orientation?

To elucidate SAB2216c's membrane topology and orientation, a multi-technique experimental approach is recommended:

• Computational prediction as starting point:

  • Analyze the SAB2216c sequence (MLYPIFIFILAGLCEIGGGYLIWLWLREGQCSLVGLIGGAILMLYGVIATFQSFPSFGRVYAAYGGVFIIMSLIFAMVVDKQMPDKYDVIGAIICIVGVLVMLLPSRA) using multiple topology prediction algorithms (TMHMM, TOPCONS, Phobius)

  • Identify potential transmembrane domains and their orientations

  • Predict membrane-interfacial regions and solvent-exposed loops

• Experimental validation methods:

  Technique	Approach	Data Interpretation
  Cysteine scanning	Introduce single Cys at various positions	Accessibility to membrane-impermeable reagents
  Protease protection	Digest proteoliposomes with specific proteases	Protected fragments indicate membrane-embedded regions
  Antibody epitope mapping	Generate antibodies to specific domains	Accessibility in intact vs. permeabilized cells
  GFP-fusion analysis	Create N and C-terminal fusions	Fluorescence quenching in acidic compartments

• Advanced structural approaches:

  • Hydrogen-deuterium exchange mass spectrometry

  • Electron paramagnetic resonance (EPR) with site-directed spin labeling

  • Cryo-electron microscopy of reconstituted protein

For rigorous validation, results from at least three independent techniques should be compared, with particular attention to potential discrepancies that might indicate dynamic topology or multiple conformational states of SAB2216c.

What approaches can be used to investigate potential post-translational modifications of SAB2216c?

To comprehensively investigate potential post-translational modifications (PTMs) of SAB2216c, researchers should implement a systematic analytical workflow:

• Mass spectrometry-based discovery approach:

  • Perform in-gel digestion using multiple proteases (trypsin, chymotrypsin) for maximum sequence coverage

  • Analyze using high-resolution LC-MS/MS with different fragmentation techniques (HCD, ETD)

  • Implement data-dependent acquisition for discovery and parallel reaction monitoring for targeted analysis

  • Search against databases with variable modifications including phosphorylation, acetylation, methylation, lipidation

• Site-specific PTM validation:

  • Generate site-specific antibodies against predicted modification sites

  • Apply chemical derivatization approaches for specific PTM types

  • Utilize metabolic labeling with PTM-specific precursors

  • Implement site-directed mutagenesis of modified residues to assess functional impact

• Temporal dynamics and regulation:

  • Compare modification patterns under different growth conditions

  • Assess PTM changes during infection models

  • Identify potential modifying enzymes through co-expression studies

  • Quantify stoichiometry of modifications at different sites

This comprehensive approach enables identification of functionally relevant modifications that might regulate SAB2216c's membrane integration, protein-protein interactions, or contribution to S. aureus physiology and virulence mechanisms.

How can researchers design experiments to assess the impact of SAB2216c on membrane permeability and antibiotic resistance?

To investigate SAB2216c's potential role in membrane permeability and antibiotic resistance, a structured experimental design with appropriate controls is essential:

• Genetic manipulation strategy:

  • Generate SAB2216c deletion mutants

  • Create controlled overexpression strains

  • Develop point mutations in predicted functional domains

  • Ensure genetic complementation controls for all mutants

• Membrane permeability assessment:

  • Implement fluorescent dye uptake assays (propidium iodide, SYTOX Green)

  • Measure leakage of intracellular components (ATP, ions)

  • Conduct electrical measurements on model membranes

  • Assess membrane potential using voltage-sensitive dyes

• Antibiotic susceptibility testing:

  Method	Measurements	Controls Required
  Broth microdilution	MIC determination for multiple antibiotics	Standard reference strains
  Time-kill kinetics	Bacterial survival over time	Growth rate normalization
  Chequerboard assays	Drug interactions (synergy/antagonism)	Single-drug controls
  Antibiotic accumulation	Uptake of labeled antibiotics	Membrane permeabilizers

• Mechanistic investigations:

  • Examine changes in membrane lipid composition

  • Assess protein-lipid interactions through lipidomic approaches

  • Investigate potential interactions with known resistance machinery

  • Quantify expression of other membrane components

This comprehensive approach allows for establishing causal relationships between SAB2216c expression, membrane properties, and antimicrobial resistance phenotypes, with implications for understanding S. aureus pathogenicity.

What experimental design considerations are important when investigating the evolution of SAB2216c across Staphylococcus species?

For evolutionary studies of SAB2216c across Staphylococcus species, a carefully structured research approach should address:

• Sequence acquisition and phylogenetic analysis:

  • Collect homologous sequences from diverse Staphylococcus species

  • Include appropriate outgroups from related genera

  • Align sequences using membrane protein-specific alignment algorithms

  • Construct phylogenetic trees using multiple methods (Maximum Likelihood, Bayesian inference)

  • Calculate evolutionary rates for different protein domains

• Structure-function relationship investigation:

  • Identify conserved vs. variable regions by mapping conservation onto predicted structure

  • Analyze selective pressure across the protein sequence (dN/dS ratios)

  • Detect potential horizontal gene transfer events

  • Correlate sequence variations with habitat differences or host specificity

• Experimental validation approach:

  Analysis Type	Method	Expected Outcome
  Functional conservation	Cross-species complementation	Rescue of deletion phenotypes
  Structural conservation	Heterologous expression and purification	Similar biochemical properties
  Localization patterns	Fluorescent protein fusions	Consistent membrane localization
  Host adaptation	Infection models with variant proteins	Species-specific differences

This integrated approach combining computational analysis with experimental validation provides insights into SAB2216c evolution and its potential role in Staphylococcus adaptation to different environmental niches or hosts.

How should researchers approach studying the effect of environmental conditions on SAB2216c expression and function?

To comprehensively investigate environmental regulation of SAB2216c, researchers should implement:

• Expression analysis under varied conditions:

  • Screen multiple environmental parameters (pH, temperature, oxygen levels, nutrient availability)

  • Test host-relevant conditions (serum exposure, antimicrobial peptides, phagocytosis)

  • Measure expression using RT-qPCR, Western blotting, and reporter fusions

  • Conduct time-course experiments to capture expression dynamics

• Promoter analysis and regulatory network identification:

  • Map the promoter region through reporter assays with truncated constructs

  • Identify potential transcription factor binding sites

  • Perform chromatin immunoprecipitation to confirm regulatory interactions

  • Screen transcription factor mutant libraries for altered SAB2216c expression

• Functional assessment under varying conditions:

  • Evaluate membrane localization changes

  • Assess protein stability and turnover rates

  • Measure functional outputs (membrane integrity, stress resistance)

  • Determine interaction partners under different conditions

This systematic approach allows for mapping the environmental responsiveness of SAB2216c expression and function, potentially revealing its role in S. aureus adaptation to different host microenvironments during infection or colonization.