Recombinant Staphylococcus aureus UPF0382 membrane protein MW0538 (MW0538)

Which expression systems are suitable for recombinant MW0538 production?

Multiple expression systems can be used for MW0538 production, each with specific advantages:

For most basic research applications, E. coli expression is sufficient for MW0538, as evidenced by successful recombinant production with N-terminal His-tag . The Creative BioMart recombinant MW0538 protein is expressed in E. coli with an N-terminal His-tag, suggesting this system provides adequate yields and proper folding for this particular membrane protein .

What purification methods are most effective for recombinant His-tagged MW0538?

For His-tagged MW0538, immobilized metal affinity chromatography (IMAC) is the primary purification method:

• Cell lysis protocol:

  • Resuspend cells in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, 1% detergent (typically DDM or LDAO for membrane proteins), and protease inhibitors

  • Disrupt cells via sonication or high-pressure homogenization

  • Centrifuge at 20,000 × g for 30 minutes to remove debris

• Membrane fraction isolation:

  • Ultracentrifuge the supernatant at 100,000 × g for 1 hour

  • Resuspend membrane pellet in solubilization buffer containing suitable detergent

• IMAC purification:

  • Load solubilized membrane fraction onto Ni-NTA or Co-NTA resin

  • Wash with increasing imidazole concentrations (20-50 mM)

  • Elute with high imidazole (250-500 mM)

• Size exclusion chromatography (SEC):

  • For higher purity, perform SEC using a Superdex 200 column

  • Use buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.03-0.05% detergent

To ensure full-length protein purification, use a sufficient imidazole concentration during elution, as this helps distinguish full-length proteins from truncated products .

What are the critical parameters for successful membrane fraction isolation of S. aureus membrane proteins?

Successful isolation of membrane fractions containing MW0538 requires careful attention to several parameters:

• Cell disruption:

  • Use gentle disruption methods (e.g., French press or sonication with cooling cycles)

  • Add protease inhibitors (PMSF, soybean trypsin inhibitor, benzamidine, trasylol) to prevent degradation

  • Maintain cold temperature (4°C) throughout the process

• Differential centrifugation:

  • Low-speed centrifugation (5,000-10,000 × g) to remove cell debris and unbroken cells

  • Ultracentrifugation (100,000 × g for 1 hour) to pellet membrane fractions

• Buffer composition:

  • Use stabilizing buffers with pH 7.4-8.0

  • Include 10-20% glycerol to enhance membrane protein stability

  • Add reducing agents (1-5 mM DTT or β-mercaptoethanol) to prevent oxidation

• Validation methods:

  • Western blot analysis using antibodies against known membrane markers

  • Enzymatic assays specific for membrane-associated functions

  • Verification of membrane thickness (5-8 nm) by atomic force microscopy

The single-cell membrane isolation ("unroofing") method described in search result demonstrates that isolated membrane patches should have a height of 5-8 nm with roughness around 1 nm when verified by AFM, which can serve as a quality control parameter for membrane preparations .

How can researchers evaluate the proper folding and functionality of recombinant MW0538?

Assessing proper folding and functionality of recombinant MW0538 involves several complementary techniques:

• Circular dichroism (CD) spectroscopy:

  • Far-UV CD (190-260 nm) to estimate secondary structure content

  • Near-UV CD (250-320 nm) to assess tertiary structure organization

  • Compare with predicted secondary structure based on sequence analysis

• Size-exclusion chromatography:

  • Monodisperse peak indicates properly folded protein

  • Aggregation or multiple peaks suggest misfolding or heterogeneity

  • Coupled with multi-angle light scattering (SEC-MALS) for absolute molecular weight determination

• Thermal stability assays:

  • Differential scanning fluorimetry (DSF) using SYPRO Orange

  • Measure melting temperature (Tm) as indicator of stability

  • Compare stability in different buffer conditions

• Functional reconstitution:

  • Incorporate purified protein into liposomes

  • Test membrane integration using flotation assays

  • Assess orientation using protease protection assays

For membrane proteins like MW0538, proper folding often correlates with detergent resistance and stability. A well-folded membrane protein should remain stable in selected detergents and not aggregate during concentration steps .

What are the state-of-the-art detergent-free methods for structural studies of MW0538?

Recent advances offer several detergent-free approaches for structural studies of membrane proteins like MW0538:

• Nanodiscs:

  • Membrane proteins are incorporated into nanometer-scale phospholipid bilayers encircled by scaffold proteins

  • Advantages: Native-like lipid environment, defined size, excellent stability

  • Protocol: Reconstitute purified MW0538 with lipids and MSP (membrane scaffold protein), followed by detergent removal via Bio-Beads

• SMALPs (Styrene Maleic Acid Lipid Particles):

  • SMA polymer extracts membrane proteins with surrounding native lipids

  • Advantages: Preserves native lipid interactions, simpler preparation

  • Limitations: pH sensitivity (requires pH >6.5), interference with certain spectroscopic methods

• NCMN (Native Cell Membrane Nanoparticles) system:

  • Uses specialized membrane-active polymers to extract proteins directly from native membranes

  • Key advantages:

    • Custom-designed polymer library for different membrane protein types

    • Protocols optimized for each NCMN polymer

    • Emphasis on native membrane environment

    • Confirmed homogeneity by electron microscopy

• Amphipols:

  • Amphipathic polymers that wrap around the hydrophobic regions of membrane proteins

  • Advantages: High stability, reduced protein aggregation

  • Applications: Electron microscopy, NMR studies, functional characterization

The NCMN system is particularly promising for MW0538 structural studies as it emphasizes working with membrane proteins in their native membrane context, which helps maintain natural protein-lipid interactions that may be critical for function .

How can single-molecule force spectroscopy (SMFS) be applied to study MW0538 structure-function relationships?

Single-molecule force spectroscopy (SMFS) enables detailed analysis of membrane protein unfolding pathways and can be applied to MW0538 using the following approach:

• Sample preparation:

  • Isolate plasma membrane fragments containing MW0538 using the "unroofing" method

  • Sandwich a single cell between two glass plates

  • Rapidly separate plates to isolate the apical membrane

  • Verify membrane patch integrity via AFM (height 5-8 nm, roughness ~1 nm)

• SMFS procedure:

  • Use non-functionalized AFM tips to avoid bias

  • Apply force to unfold proteins, generating force-distance (F-D) curves

  • Select curves showing sawtooth patterns characteristic of protein unfolding

  • Fit peaks to the worm-like chain (WLC) model with persistence length ~0.4 nm

• Data analysis:

  • Perform unsupervised clustering to identify similar unfolding patterns

  • Apply Bayesian meta-analysis using protein structure databases

  • Compare experimental data with predicted unfolding patterns based on MW0538 topology

• Validation:

  • Express MW0538 with specific modifications at key domains

  • Compare unfolding signatures to confirm protein identification

  • Correlate unfolding patterns with functional domains

This methodology has successfully characterized over 40 membrane protein unfolding spectra and identified four mammalian membrane proteins, suggesting it could be applied to study MW0538 in its native environment .

What approaches can resolve contradictory experimental data regarding MW0538 topology and orientation?

When faced with contradictory data regarding MW0538's membrane topology and orientation, a systematic approach combining multiple techniques is recommended:

• Computational prediction comparison:

  • Apply multiple topology prediction algorithms (TMHMM, HMMTOP, MEMSAT, etc.)

  • Create consensus models from overlapping predictions

  • Generate a topology map showing agreement/disagreement between methods

• Experimental validation techniques:

  Technique	Principle	Resolution	Limitations
  Cysteine scanning	Introduce single cysteines at various positions; assess accessibility to membrane-impermeable reagents	Residue level	Requires cysteine-free background
  GFP fusion analysis	Fusion of GFP to different protein segments; fluorescence indicates cytoplasmic localization	Domain level	May disrupt protein folding
  Proteolytic digestion	Limited proteolysis of accessible regions; MS identification of protected fragments	Domain level	Incomplete digestion complications
  FRET analysis	Measure distances between labeled residues to validate structural models	Angstrom level	Requires specific labeling

• Reconstitution studies:

  • Insert purified MW0538 into liposomes with defined orientation

  • Use fluorescent probes to determine sidedness

  • Correlate orientation with functional assays

• Cross-validation strategy:

  • Integrate data from all methods to build comprehensive model

  • Weight evidence based on method reliability

  • Test predictions with site-directed mutagenesis of key residues

Researchers have successfully resolved contradictory data for other membrane proteins by combining in silico predictions with targeted experimental approaches, particularly the cysteine accessibility method which can provide residue-level topology information .

What methods are most suitable for investigating MW0538 protein-protein interactions in the S. aureus membrane?

Investigating MW0538's protein-protein interactions requires techniques that preserve membrane context:

• In vivo crosslinking approaches:

  • Chemical crosslinking with membrane-permeable reagents (DSP, formaldehyde)

  • Photo-activated crosslinking using genetically incorporated unnatural amino acids

  • Analysis by MS to identify crosslinked partners

• Co-immunoprecipitation (Co-IP) adaptations:

  • Solubilize membranes with mild detergents (DDM, digitonin)

  • Use anti-MW0538 antibodies or anti-tag antibodies for pulldown

  • Confirm interactions by two-way Co-IP as demonstrated for other membrane proteins

  • Identify partners by Western blot or MS analysis

• Proximity-based labeling:

  • Fusion of MW0538 with BioID or APEX2 enzymes

  • Biotin labeling of proximal proteins in living cells

  • Streptavidin pulldown and MS identification of biotinylated proteins

• Membrane-based yeast two-hybrid systems:

  • Split-ubiquitin membrane yeast two-hybrid (MYTH)

  • Bait-prey screening against S. aureus genomic library

  • Validation of positives with quantitative β-galactosidase assays

An integrated approach combining crosslinking with co-immunoprecipitation has been successful for other membrane proteins, as demonstrated in the two-way co-immunoprecipitation studies detailed in search result , which confirmed protein interactions by performing reciprocal pulldowns from both protein partners .

How can researchers design assays to determine the specific function of MW0538 in S. aureus?

To determine the specific function of MW0538, researchers should implement a multi-faceted approach:

• Genetic manipulation strategies:

  • Generate MW0538 knockout strains using CRISPR-Cas9 or allelic replacement

  • Create conditional expression strains (inducible promoters)

  • Evaluate phenotypic changes in growth, membrane integrity, and stress response

• Functional complementation:

  • Reintroduce wild-type or mutant variants of MW0538 to knockout strains

  • Assess restoration of phenotypes

  • Test heterologous expression in model organisms

• Comparative genomics approach:

  • Identify homologs in related species

  • Analyze conservation patterns and co-evolution with other genes

  • Predict function based on genomic context and conserved domains

• Phenotypic microarray analysis:

  • Expose wild-type and MW0538-deficient strains to diverse growth conditions

  • Monitor responses to different carbon sources, pH values, and antibiotics

  • Identify conditions where MW0538 provides advantage or disadvantage

A parallel approach used for similar membrane proteins involved studying growth kinetics in different stress conditions (osmotic, oxidative, antibiotic) and membrane integrity assays using fluorescent dyes like propidium iodide or SYTOX green to assess membrane permeability differences between wild-type and knockout strains .

What role might MW0538 play in S. aureus virulence and pathogenicity?

To explore MW0538's potential role in S. aureus virulence:

• Infection model systems:

  • Compare wild-type and MW0538-deficient strains in:

    • Cell culture infection models (adhesion, invasion, intracellular survival)

    • Simple animal models (Galleria mellonella, Caenorhabditis elegans)

    • Mammalian infection models (mouse sepsis, pneumonia, skin infection)

  • Measure bacterial burden, dissemination, and host response markers

• Virulence factor expression analysis:

  • Examine expression of known virulence factors in MW0538 mutants:

    • Toxins (α-hemolysin, enterotoxins)

    • Surface proteins (protein A, IsdB)

    • Regulatory systems (agr, sae)

  • Use qRT-PCR, Western blot, and reporter gene assays

• Host-pathogen interaction studies:

  • Assess MW0538 mutant interaction with host immune components:

    • Phagocytosis by neutrophils and macrophages

    • Complement activation and deposition

    • Inflammatory cytokine responses

• Comparative virulence profiling:

  • Test MW0538 expression levels across:

    • Clinical vs. laboratory strains

    • Antibiotic-resistant vs. sensitive isolates

    • Invasive vs. colonizing isolates

While MW0538's specific role has not been directly described, other S. aureus membrane proteins have been identified as potential vaccine candidates. For example, research on a five-antigen S. aureus vaccine (rFSAV) demonstrated that targeting conserved membrane antigens could provide protection in mouse models of lethal sepsis and pneumonia . This suggests that membrane proteins like MW0538 might have immunogenic properties or roles in bacterial survival during infection.

How can researchers overcome common challenges in expressing full-length MW0538?

Expression of full-length membrane proteins like MW0538 presents several challenges. Here are evidence-based solutions:

• Protein toxicity issues:

  • Use tightly regulated expression systems (T7lac, arabinose-inducible)

  • Reduce induction temperature (16-25°C)

  • Lower inducer concentration and extend expression time

  • Consider specialized E. coli strains (C41/C43, Lemo21)

• Truncated product formation:

  • Optimize translation initiation sites to minimize internal translation

  • Use dual affinity tags (N- and C-terminal) to purify only full-length protein

  • Increase imidazole concentration during elution to distinguish full-length from truncated products

  • Add protease inhibitors during all purification steps

• Inclusion body formation:

  • Reduce expression rate by lowering temperature and inducer concentration

  • Co-express molecular chaperones (GroEL/ES, DnaK/J)

  • Test fusion partners that enhance solubility (MBP, SUMO, thioredoxin)

  • Consider refolding protocols if inclusion bodies are unavoidable

• Membrane integration challenges:

  • Ensure proper signal sequence is present

  • Co-express translocation machinery components if needed

  • Add mild detergents during cell lysis to improve extraction

For MW0538 specifically, research indicates that E. coli expression with an N-terminal His-tag has been successful when expressed at reduced temperatures (18-20°C) with moderate IPTG concentrations (0.2-0.5 mM) .

What are the critical factors for maintaining stability of purified MW0538 during structural and functional studies?

Maintaining stability of purified MW0538 requires careful attention to several factors:

• Buffer optimization:

  Component	Recommended Range	Purpose
  pH	7.0-8.0	Maintain protein stability
  Salt	150-300 mM NaCl	Reduce nonspecific interactions
  Glycerol	5-10%	Enhance stability
  Reducing agent	1-5 mM DTT or TCEP	Prevent oxidation
  Detergent	CMC + 0.05%	Maintain protein solubility

• Detergent selection criteria:

  • Screen multiple detergents (DDM, LDAO, DM, OG)

  • Assess stability using thermal shift assays

  • Consider detergent mixtures or facial amphiphiles

  • Test cholesterol or specific lipid addition

• Storage conditions optimization:

  • Test stability at different temperatures (4°C, -20°C, -80°C)

  • Evaluate freeze-thaw stability (avoid multiple cycles)

  • Consider flash-freezing in liquid nitrogen

  • Add stabilizing agents (trehalose, sucrose)

• Handling precautions:

  • Minimize concentration steps to avoid aggregation

  • Use low-binding tubes and filters

  • Maintain temperature consistency during experiments

  • Centrifuge before use to remove potential aggregates

For membrane proteins like MW0538, storage in Tris/PBS-based buffer with 6% trehalose at pH 8.0 has been shown to maintain stability. After reconstitution, adding glycerol (final concentration 5-50%) and aliquoting for long-term storage at -20°C/-80°C is recommended to avoid repeated freeze-thaw cycles .

How can researchers address data reproducibility challenges in MW0538 structural studies?

Addressing reproducibility challenges in structural studies of membrane proteins like MW0538 requires systematic approaches:

• Standardization of protein preparation:

  • Develop detailed standard operating procedures (SOPs)

  • Implement quality control checkpoints (purity, homogeneity, activity)

  • Use reference standards to calibrate purification methods

  • Document batch variation with analytical techniques (SEC, DLS, MS)

• Validation through orthogonal methods:

  • Compare results from multiple structural techniques:

    • X-ray crystallography

    • Cryo-electron microscopy

    • NMR spectroscopy

    • SMFS (single-molecule force spectroscopy)

  • Evaluate consistency across different sample preparations

• Rigorous statistical analysis:

  • Increase sample size and biological replicates

  • Use appropriate statistical tests for significance

  • Implement Bayesian analysis frameworks as described for SMFS data

  • Report all experimental attempts, not just successful ones

• Environmental variable control:

  • Standardize buffer components and pH

  • Control temperature during all experiments

  • Document equipment calibration and maintenance

  • Consider automated sample handling to reduce operator variability

The approach described in search result demonstrates how researchers addressed reproducibility in single-molecule force spectroscopy by implementing a Bayesian framework that combines experimental data with information from mass spectrometry and protein structure databases, allowing more reliable identification of membrane proteins .

What emerging technologies might advance our understanding of MW0538 structure and function?

Several cutting-edge technologies show promise for advancing MW0538 research:

These advanced approaches could significantly enhance our understanding of MW0538's structure-function relationships within the context of the S. aureus membrane environment .

How might MW0538 research contribute to novel antimicrobial strategies against S. aureus?

Research on MW0538 could contribute to antimicrobial development through several avenues:

• Vaccine development potential:

  • Assessment as vaccine antigen candidate:

    • Evaluate conservation across S. aureus strains

    • Test immunogenicity and protective efficacy

    • Consider as component of multi-antigen vaccines

  • The success of the five-antigen S. aureus vaccine (rFSAV) targeting conserved antigens suggests membrane proteins can be effective vaccine components

• Small molecule inhibitor design:

  • Structure-based drug design:

    • Identify druggable pockets in MW0538 structure

    • Virtual screening of compound libraries

    • Fragment-based approaches to develop selective inhibitors

  • Target validation through genetic and pharmacological methods

• Antibody-based therapeutics:

  • Development of antibodies targeting exposed epitopes

  • Antibody-antibiotic conjugates for targeted delivery

  • Bispecific antibodies linking MW0538 with immune effectors

• Membrane disruption strategies:

  • Peptides designed to interact with MW0538 and disrupt membrane integrity

  • Nanoparticles targeting MW0538-enriched membrane domains

  • Exploiting MW0538's potential role in maintaining membrane homeostasis

Research on other S. aureus membrane proteins has shown that targeting conserved virulence factors can provide broad protection against diverse strains, suggesting similar approaches could be valuable if MW0538 plays a role in pathogenesis .

What unexplored aspects of MW0538 biology warrant further investigation?

Several unexplored aspects of MW0538 biology offer promising research opportunities:

• Evolutionary significance:

  • Comparative genomics across Staphylococcal species

  • Analysis of selection pressure on different protein domains

  • Investigation of horizontal gene transfer events

  • Correlation with bacterial adaptation to different ecological niches

• Regulatory networks:

  • Transcriptional regulation under different stress conditions

  • Post-translational modifications affecting function

  • Integration in quorum sensing networks

  • Role in biofilm formation and maintenance

• Lipid interactions:

  • Specific lipid requirements for function

  • Influence on local membrane curvature or thickness

  • Association with lipid rafts or functional membrane domains

  • Impact of host-derived lipids during infection

• Structural dynamics:

  • Conformational changes in response to environmental stimuli

  • Oligomerization states under different conditions

  • Allosteric regulation mechanisms

  • Protein dynamics in native membrane environment

• Host-pathogen interface:

  • Potential interactions with host receptors or immune components

  • Role in evading host defense mechanisms

  • Contribution to bacterial persistence or antibiotic tolerance

  • Function during different stages of infection