Recombinant Staphylococcus aureus UPF0060 membrane protein MW2259 (MW2259)

Optimal Expression Systems

Q: Which expression system provides the highest yield of functional MW2259 protein?

A: For recombinant S. aureus membrane proteins like MW2259, modified E. coli BL21(DE3) strains with deletions in major outer membrane proteins have demonstrated significant improvements in expression yield and quality. Research has shown that quadruple mutant strains (BL21ΔABCF) with four major OMPs deleted exhibit enhanced levels of membrane protein expression compared to the parent BL21(DE3) strain . When expressing MW2259, consider using these specialized strains that have been engineered to accommodate additional membrane proteins with reduced competition for membrane insertion machinery.

Q: What induction parameters maximize MW2259 expression while preserving protein structure?

A: Optimal induction conditions for S. aureus membrane proteins involve moderate temperatures and controlled inducer concentrations. Evidence suggests inducing cultures at OD600 ~0.5 with either isopropyl thiogalactoside (1 mM) or anhydrotetracycline (50 ng/ml), followed by incubation at 30°C for 2-3 hours . For MW2259 specifically, lower temperatures (25-30°C) may be beneficial as they slow expression rates, allowing proper membrane insertion. Growth characteristics of expression strains may vary significantly from parent strains, with BL21ΔABCF growing more slowly than BL21(DE3), so adjust protocols accordingly .

Advanced Purification Approaches

Q: How can I optimize membrane fraction isolation to maintain MW2259 in its native conformation?

A: For optimal isolation of MW2259 in its native conformation, a sequential fractionation approach is recommended. Begin with gentle cell disruption methods (French press or sonication) in a buffer containing 20 mM Tris-HCl pH 7.4, 150 mM NaCl, and protease inhibitors. Separate membrane fractions through differential centrifugation, first clearing cell debris (10,000 × g, 20 min) followed by ultracentrifugation (100,000 × g, 1 hour) to pellet membranes. Research indicates that membrane protein-enriched extracellular vesicles (MPEEVs) provide an excellent platform for studying intact membrane proteins with correct topology , which may be applicable to MW2259 isolation while preserving native structure.

Q: What detergent screening strategy is most effective for solubilizing MW2259?

A: Implement a systematic detergent screening approach using the panel outlined in Table 1. Begin with mild detergents such as DDM at 1-2% for initial solubilization, then buffer exchange to maintenance concentrations (0.05-0.1%). Research with similar membrane proteins suggests a two-detergent system may provide optimal results - using a stronger detergent for initial extraction and a milder one for long-term stability .

Table 1: Detergent Screening Panel for MW2259 Solubilization

Primary Structure Analysis

Q: How can I verify the complete sequence of recombinant MW2259 protein?

A: Complete sequence verification requires combining multiple analytical approaches. Begin with tryptic digestion followed by LC-MS/MS peptide mapping, which can typically achieve >90% sequence coverage. Areas with poor coverage, particularly hydrophobic transmembrane regions, should be analyzed using alternative proteases like chymotrypsin or pepsin. For N-terminal sequencing, Edman degradation remains valuable for confirming the precise start of the mature protein. Alternative techniques like top-down mass spectrometry can analyze the intact protein to identify potential post-translational modifications. When analyzing MS data, carefully compare observed versus theoretical masses to identify any unexpected modifications that may have occurred during expression or purification.

Membrane Protein Structure Determination

Q: What approaches are most suitable for determining the structure of MW2259 in its membrane environment?

A: For membrane proteins like MW2259, a multi-technique approach yields the most comprehensive structural information. Table 2 compares the primary methods with their advantages for membrane protein analysis.

Table 2: Comparison of Structural Analysis Methods for MW2259

Research indicates that membrane protein-enriched extracellular vesicles (MPEEVs) provide a promising platform for studying intact membrane proteins with native anchoring and correct topology , which could be particularly valuable for MW2259 structural studies.

Conformational Analysis Techniques

Q: How can I monitor dynamic conformational changes in MW2259 during its functional cycle?

A: Tracking dynamic behavior of MW2259 requires specialized biophysical approaches. Single-molecule Förster Resonance Energy Transfer (smFRET) can reveal distance changes between strategically labeled positions in the protein. Site-specific labeling at cysteine residues introduced through mutagenesis allows precise placement of fluorophores for detecting conformational shifts during substrate binding or transport cycles. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) provides complementary data by identifying regions with altered solvent accessibility during functional state transitions. For direct visualization in lipid environments, high-speed atomic force microscopy (HS-AFM) can capture conformational changes with nanometer resolution in real-time . Integrating these approaches provides a comprehensive picture of MW2259's dynamic structural landscape.

Host-Pathogen Interaction Studies

Q: What methodologies can identify potential interactions between MW2259 and host immune components?

A: Investigating MW2259's role in host-pathogen interactions requires multiple complementary approaches. Research has shown that S. aureus proteins can activate human monocytes, leading to expression of immune stimulatory natural killer group 2D (NKG2D) ligands . To determine if MW2259 participates in similar immune modulation, implement the workflow in Table 3.

Table 3: Workflow for Identifying MW2259-Host Immune Interactions

Q: How can I determine if MW2259 contributes to S. aureus immune evasion mechanisms?

A: Recent research indicates that S. aureus has evolved specific mechanisms to modulate host immunity, including the regulation of NKG2D ligand expression . To assess MW2259's potential role in immune evasion, compare wild-type S. aureus with MW2259 knockout and complemented strains in immune recognition assays. Investigate whether MW2259 affects phagocytosis rates, complement deposition, or antimicrobial peptide resistance. Research has shown that S. aureus ClpP protease functionality influences ULBP2 induction and proinflammatory cytokine production in human monocytes , suggesting potential protease-dependent pathways that may involve membrane proteins like MW2259. Additionally, examine whether MW2259 participates in biofilm formation, which can serve as a physical barrier against immune detection.

Metabolic and Physiological Impact

Q: How does MW2259 expression affect bacterial metabolism and stress responses?

A: To assess MW2259's metabolic impact, implement comparative metabolomic and physiological analyses between wild-type S. aureus and MW2259 mutant strains. Research has shown that S. aureus proteins can influence cell metabolism, including changes in cytoplasmic (iso)citrate levels, glycolytic flux, and mitochondrial activity . For MW2259, conduct LC-MS metabolomics to identify altered metabolite profiles under various growth conditions. Measure oxygen consumption rates, membrane potential, and ATP production to assess bioenergetic consequences of MW2259 expression or deletion. RNA-sequencing of these strains can reveal compensatory transcriptional changes in metabolic and stress response genes. For comprehensive analysis, stable isotope labeling experiments can track specific metabolic fluxes and identify precisely which pathways are affected.

Q: What role might MW2259 play in S. aureus adaptation to different host environments?

A: Investigating MW2259's role in host adaptation requires comparative analysis across different conditions. Research has shown that S. aureus undergoes recombination-mediated remodeling to adapt to specific niches, affecting genes involved in host-pathogen interactions . To determine if MW2259 contributes to this adaptation, compare expression levels across S. aureus strains isolated from different host species or infection sites. Examine MW2259 sequence conservation and variation across these isolates to identify potential host-specific adaptations. Challenge wild-type and MW2259 mutant strains with conditions mimicking different host microenvironments (varying pH, oxygen levels, nutrient availability) and measure survival rates. These experiments will help determine whether MW2259 functions as part of S. aureus' adaptive toolkit for colonizing diverse host niches.

Expression and Purification Troubleshooting

Q: How can I resolve poor membrane insertion when expressing recombinant MW2259?

A: Poor membrane insertion of MW2259 requires systematic optimization. Research shows that modified E. coli strains with deletions in major outer membrane proteins (BL21ΔABCF) can significantly improve membrane insertion of recombinant proteins . Evidence demonstrates improved expression of test proteins in this quadruple mutant strain compared to the parent BL21(DE3), visible through both Coomassie staining and Western blotting . Additional strategies include reducing expression temperature (16-20°C), decreasing inducer concentration, and using slower induction methods. Consider fusion partners that enhance membrane targeting, such as Mistic or YidC. For verification of proper membrane insertion, implement a fractionation control experiment comparing cytoplasmic, inclusion body, and membrane fractions using Western blotting with anti-MW2259 antibodies.

Q: What approaches can overcome protein aggregation during MW2259 purification?

A: Addressing MW2259 aggregation requires a multi-faceted approach focusing on maintaining the protein's native environment. First, optimize detergent selection through systematic screening (see Table 1), as inappropriate detergents are a primary cause of membrane protein aggregation. Include stabilizing additives in purification buffers: glycerol (10-20%), cholesteryl hemisuccinate (0.01-0.05%), and specific lipids like phosphatidylglycerol that mimic the S. aureus membrane environment. Maintain strict temperature control during purification, keeping samples at 4°C throughout. Implement size exclusion chromatography as a final purification step to separate aggregates and monitor monodispersity. Research with other membrane proteins demonstrates that these approaches can significantly reduce aggregation while maintaining functional integrity .

Structural Integrity Verification

Q: How can I confirm that detergent-solubilized MW2259 maintains its native structure?

A: Validating the native structure of detergent-solubilized MW2259 requires multiple complementary approaches. Circular dichroism spectroscopy can confirm secondary structure content matches predictions for this membrane protein. Thermal stability assays using differential scanning fluorimetry help compare protein stability across different detergent conditions, identifying those that best maintain structural integrity. Research indicates that membrane protein-enriched extracellular vesicles (MPEEVs) can provide a reference for native membrane protein structure . For functional validation, develop activity assays specific to MW2259's function and compare activity in detergent with that in native membrane extracts or reconstituted proteoliposomes. Advanced techniques like hydrogen-deuterium exchange mass spectrometry can map solvent-protected regions, which should correspond to transmembrane segments in properly folded protein.

Q: What lipid reconstitution methods best preserve MW2259 function for in vitro studies?

A: For optimal functional reconstitution of MW2259, select lipid compositions mimicking the S. aureus membrane environment. A recommended protocol involves solubilizing a mixture of POPE:POPG (7:3) lipids with mild detergents like DDM, combining with purified protein at 1:100 protein:lipid ratio, then removing detergent using Bio-Beads SM-2 or controlled dialysis. For functional studies, proteoliposomes offer a simplified environment, while nanodiscs formed using membrane scaffold proteins provide a more native-like bilayer with defined size. Recent advances demonstrate that reconstitution into membrane protein-enriched extracellular vesicles (MPEEVs) can maintain proteins in their native orientation with correct topology . This approach has proven particularly valuable for structural and functional studies of challenging membrane proteins.

Evolutionary and Comparative Analysis

Q: How can comparative genomics inform functional studies of MW2259 across S. aureus strains?

A: Comparative genomic analysis can reveal critical insights into MW2259 function and evolution. Research has demonstrated that S. aureus undergoes recombination-mediated remodeling that affects genes involved in host-pathogen interactions . For MW2259, begin by compiling sequences from diverse S. aureus isolates across different host species, geographic regions, and clinical outcomes. Analyze sequence conservation patterns to identify highly conserved regions likely essential for function versus variable regions that may contribute to host adaptation. Pay particular attention to strains that have undergone large-scale recombination events, as research shows these can lead to acquisition of novel pathogenic traits . Construct phylogenetic trees based on MW2259 sequences to identify potential horizontal gene transfer events. These analyses can guide functional studies by highlighting regions of interest for targeted mutagenesis or chimeric protein construction.

Q: What can structural homology modeling reveal about MW2259 function?

A: Structural homology modeling provides valuable insights when experimental structures are unavailable. Begin by identifying structural homologs using tools like HHpred or I-TASSER, focusing on membrane proteins with similar topology rather than sequence identity alone. Even distant homologs can reveal functional motifs and potential ligand-binding sites. Once models are generated, evaluate quality using metrics like QMEAN and ProCheck to identify reliable regions. Use molecular dynamics simulations in a lipid bilayer environment to refine models and predict stable conformations. Research on other membrane proteins demonstrates that integrating evolutionary information with structural predictions can identify functional interfaces . Analyze surface conservation patterns mapped onto the model to predict protein-protein interaction sites or substrate-binding pockets, generating testable hypotheses for experimental validation.

Emerging Methodologies

Q: How can advanced imaging techniques enhance our understanding of MW2259 localization and dynamics?

A: Cutting-edge imaging approaches offer unprecedented insights into MW2259 behavior in its native context. Super-resolution microscopy techniques like PALM/STORM can visualize MW2259 distribution in bacterial membranes with 20-30 nm resolution. For dynamic studies, implement single-particle tracking with photoactivatable fluorescent proteins fused to MW2259 to monitor diffusion dynamics and potential clustering behavior. Cryo-electron tomography provides structural context by visualizing MW2259 within the bacterial membrane landscape at molecular resolution. Research has demonstrated that membrane protein-enriched extracellular vesicles (MPEEVs) provide an excellent platform for such studies , allowing visualization of correctly oriented membrane proteins. For highest resolution, correlative light and electron microscopy (CLEM) can precisely locate fluorescently-tagged MW2259 within cellular ultrastructure.

Q: What are the advantages of cell-free expression systems for structural studies of MW2259?

A: Cell-free expression systems offer distinct advantages for MW2259 structural studies. These systems bypass toxicity issues often encountered with membrane protein overexpression in living cells . Using E. coli lysate supplemented with nanodiscs or lipid-detergent mixtures enables direct incorporation of MW2259 into membrane mimetics during translation, avoiding aggregation. This approach allows rapid screening of different detergents and lipids to identify optimal conditions for structural stability. Another significant advantage is the ability to incorporate unnatural amino acids at specific positions, enabling site-specific labeling for biophysical studies or creating heavy atom derivatives for crystallography. For NMR studies, cell-free systems excel at selective isotope labeling of specific amino acid types, simplifying spectral assignment of complex membrane proteins like MW2259.