Recombinant Staphylococcus aureus UPF0060 membrane protein USA300HOU_2320 (USA300HOU_2320)

What is Recombinant Staphylococcus aureus UPF0060 membrane protein USA300HOU_2320?

Recombinant Staphylococcus aureus UPF0060 membrane protein USA300HOU_2320 is a full-length (108 amino acids) bacterial membrane protein originally derived from Staphylococcus aureus but recombinantly expressed in E. coli expression systems. The protein features an N-terminal histidine tag to facilitate purification and detection. The amino acid sequence is: MLYPIFIFILAGLCEIGGGYLIWLWLREGQSSLVGLIGGAILMLYGVIATFQSFPSFGRVYAAYGGVFIIMSLIFAMVVDKQMPDKYDVIGAIICIVGVLVMLLPSRA . This protein belongs to the UPF0060 family, which consists of uncharacterized membrane proteins with predicted functions in cellular transport or signaling pathways. The USA300 designation indicates its association with a particular strain of Staphylococcus aureus that has been implicated in community-acquired methicillin-resistant S. aureus (CA-MRSA) infections .

What are the optimal storage conditions for USA300HOU_2320 protein?

For optimal stability and activity retention of recombinant USA300HOU_2320 protein, researchers should adhere to the following evidence-based storage protocol:

• Store the lyophilized powder at -20°C to -80°C upon receipt

• Perform aliquoting immediately after reconstitution to prevent protein degradation from repeated freeze-thaw cycles

• For working stocks, maintain aliquots at 4°C for up to one week

• For long-term storage, add glycerol to a final concentration of 5-50% (with 50% being optimal) and store at -20°C to -80°C

It's recommended to briefly centrifuge the vial before opening to ensure all material is at the bottom of the tube. After reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL, the protein should be stored in a Tris/PBS-based buffer containing 6% Trehalose at pH 8.0 to maintain stability .

How should USA300HOU_2320 protein be reconstituted for experimental use?

Proper reconstitution of USA300HOU_2320 is critical for maintaining protein functionality in experimental applications. Follow this methodological approach:

• Centrifuge the vial briefly before opening to bring contents to the bottom

• Reconstitute the lyophilized protein in deionized sterile water to achieve a final concentration between 0.1-1.0 mg/mL

• For enhanced stability, add glycerol to a final concentration of 5-50% (50% is recommended as standard)

• Aliquot the reconstituted protein immediately to prevent degradation from repeated freeze-thaw cycles

• Verify protein concentration using standardized methods such as Bradford assay or BCA

The reconstituted protein should appear clear without visible precipitation. If aggregation is observed, gentle vortexing or pipetting may help disperse protein clusters. For applications requiring higher purity, researchers should verify protein quality via SDS-PAGE, which should demonstrate >90% purity for this recombinant preparation .

What experimental techniques are most suitable for studying USA300HOU_2320?

The membrane protein USA300HOU_2320 can be studied using multiple complementary experimental approaches:

• Structural Analysis: Techniques such as X-ray crystallography, cryo-electron microscopy, or NMR spectroscopy can elucidate the three-dimensional structure, particularly useful given its relatively small size (108 amino acids).

• Functional Assays: Due to its membrane localization, liposome reconstitution assays or black lipid membrane experiments can assess potential transport or channel functions.

• Protein-Protein Interaction Studies: Pull-down assays utilizing the His-tag, co-immunoprecipitation, or yeast two-hybrid screening can identify interaction partners within the bacterial membrane or cytoplasm.

• Expression Analysis: qPCR and Western blotting can monitor expression levels under various conditions, such as antibiotic exposure or environmental stressors.

• Localization Studies: Immunofluorescence microscopy or fractionation studies can confirm membrane localization and distribution patterns within bacterial cells.

These techniques should be selected based on specific research questions, with experimental designs incorporating true experimental research principles including appropriate controls and randomization to ensure reliable and reproducible results .

How can USA300HOU_2320 be effectively incorporated into experimental designs studying antibiotic resistance?

To investigate potential roles of USA300HOU_2320 in antibiotic resistance mechanisms, researchers should implement a multi-faceted experimental approach:

• Gene Expression Correlation Studies: Design experiments comparing USA300HOU_2320 expression levels in susceptible versus resistant S. aureus strains using RT-qPCR under standardized conditions. This should involve a true experimental research design with proper randomization and controls .

• Knockout/Knockdown Phenotype Analysis: Create gene deletion mutants or implement CRISPR-Cas9 knockdown systems to evaluate changes in minimum inhibitory concentration (MIC) profiles across multiple antibiotic classes.

• Protein-Antibiotic Interaction Assays: Utilize surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) with the purified recombinant protein to detect direct binding between USA300HOU_2320 and antibiotic compounds.

• Membrane Permeability Assessments: Compare membrane integrity and permeability in wild-type versus USA300HOU_2320-modified strains using fluorescent dyes or radioactively labeled antibiotics.

• Structural Modeling: Develop computational models predicting potential antibiotic binding sites or membrane topology alterations that might influence resistance mechanisms.

These approaches should incorporate proper statistical design principles following a Solomon four-group design where appropriate to control for potential confounding variables . Researchers should track relevant strain information, as MRSA has a complex evolutionary history with multiple resistance mechanisms that may interact with membrane protein functions .

What are the challenges in expressing USA300HOU_2320 for structural studies, and how can they be overcome?

Membrane proteins like USA300HOU_2320 present significant challenges for structural studies due to their hydrophobic nature. Researchers should consider these methodological solutions:

• Expression System Optimization:

  • While E. coli is the standard expression system , alternative hosts such as Pichia pastoris or insect cells may provide better folding environments for membrane proteins

  • Implement inducible expression systems with fine-tuned promoter strength to prevent toxicity from overexpression

  • Consider fusion partners beyond the His-tag, such as MBP or SUMO, to enhance solubility

• Detergent Selection:

  • Screen multiple detergents (DDM, LDAO, OG) for optimal extraction efficiency

  • Implement detergent exchange procedures during purification to identify stabilizing conditions

  • Consider amphipols or nanodiscs for maintaining native-like membrane environments

• Crystallization Strategies:

  • Lipidic cubic phase (LCP) crystallization may be more effective than traditional vapor diffusion methods

  • In situ crystallization screening with automated imaging systems

  • Surface entropy reduction mutations to promote crystal contact formation

• Alternative Structural Methods:

  • Cryo-EM single-particle analysis for detergent-solubilized protein

  • Solid-state NMR for membrane-embedded protein structure

  • SAXS/SANS for low-resolution envelope determination

Researchers should implement a systematic approach to expression optimization, tracking protein quality at each step using techniques like SEC-MALS, thermal shift assays, and functional validation to ensure that the recombinant protein maintains native conformational properties.

How can USA300HOU_2320 be studied in spaceflight experiments to understand its role in Staphylococcus aureus adaptations?

Spaceflight experiments offer unique opportunities to understand bacterial adaptation mechanisms in microgravity environments, which may provide insights into stress responses relevant to antimicrobial resistance. Based on methodologies from previous space-based microbiology experiments, researchers should consider:

• Hardware Selection and Validation:

  • Use specialized containment systems validated for spaceflight conditions

  • Develop passive temperature control strategies using validated materials like ICE Bricks (10-12°C) for sample preservation during transit

  • Implement oxygen-permeable growth systems to avoid hypoxic conditions that can compromise experiments

• Experimental Design Considerations:

  • Prepare control experiments on Earth under matched conditions

  • Design experiments to accommodate potential launch delays (24-48 hours) based on viability testing

  • Include automated fixation or sample processing capabilities to capture time-dependent changes

• Analytical Approaches:

  • Transcriptomic analysis to measure USA300HOU_2320 expression changes in microgravity

  • Proteomic profiling to detect post-translational modifications or abundance changes

  • Membrane fluidity assessments to determine structural adaptations in the bacterial membrane

• Data Analysis Framework:

  • Implement matched population thresholds (>45,000 cells per sample) to ensure sufficient material for post-flight analysis

  • Develop comparative analysis workflows between flight and ground control samples

  • Correlate USA300HOU_2320 expression patterns with phenotypic changes in antibiotic susceptibility

Researchers should validate experimental timings and conditions through ground-based simulations before actual spaceflight implementation, accounting for the unique challenges of conducting biological research in space .

What protein-protein interaction networks might involve USA300HOU_2320, and how can they be experimentally mapped?

Mapping the protein-protein interaction network for USA300HOU_2320 requires a multi-method approach:

• Proximity-based Labeling:

  • BioID or APEX2 fusion constructs to identify proximal proteins in the native membrane environment

  • Crosslinking mass spectrometry (XL-MS) to capture direct binding partners

  • Split protein complementation assays to validate specific interactions

• Systems-level Approaches:

  • Bacterial two-hybrid screening against comprehensive S. aureus genomic libraries

  • Co-immunoprecipitation followed by mass spectrometry (Co-IP-MS)

  • Quantitative SILAC proteomics comparing wild-type and USA300HOU_2320 knockout strains

• Bioinformatic Prediction and Validation:

  • Use structural homology and co-expression patterns to predict interaction partners

  • Implement network analysis algorithms to identify hub proteins and functional modules

  • Validate predictions with targeted binary interaction assays

• Functional Correlation Studies:

  • Epistasis analysis between USA300HOU_2320 and candidate genes

  • Co-localization studies using fluorescently tagged proteins

  • Phenotypic convergence analysis of respective mutants

This comprehensive mapping approach would help place USA300HOU_2320 within the broader context of S. aureus biology, potentially revealing its role in virulence, antibiotic resistance, or membrane homeostasis pathways .

What are the optimal conditions for using recombinant USA300HOU_2320 protein in antimicrobial screening assays?

When designing antimicrobial screening assays incorporating USA300HOU_2320 protein, researchers should optimize conditions according to these methodological guidelines:

• Buffer Composition:

  • Maintain protein in Tris/PBS-based buffer at pH 8.0 with 6% trehalose for stability

  • Include appropriate detergent concentrations (0.01-0.05% DDM) to prevent aggregation

  • Consider ionic strength effects on protein-compound interactions (150-300 mM NaCl recommended)

• Protein Concentration Determination:

  • Use standardized protein quantification methods (BCA or Bradford assays)

  • Validate activity using functional assays before screening

  • Maintain protein concentrations between 0.1-1.0 mg/mL for most assay formats

• Assay Format Selection:

  • Liposome reconstitution for transport assays

  • Surface plasmon resonance for direct binding studies

  • Thermal shift assays for compound binding validation

• Control Implementation:

  • Include known membrane protein inhibitors as positive controls

  • Use denatured protein preparations as negative controls

  • Implement statistical validation through Z-factor determination

• Data Analysis Framework:

  • Apply dose-response modeling for hit compounds

  • Implement correction factors for compound intrinsic fluorescence or aggregation

  • Cross-validate hits through orthogonal assay formats

Researchers should conduct preliminary validation experiments to determine assay reproducibility and sensitivity before proceeding to large-scale screening, following true experimental research design principles with appropriate randomization and controls .

How can researchers distinguish between functional effects of USA300HOU_2320 and experimental artifacts in membrane studies?

Distinguishing genuine functional effects from artifacts when studying membrane proteins requires rigorous control experiments and methodological considerations:

• Expression Level Control:

  • Implement tunable expression systems to test dose-dependent effects

  • Create calibration curves relating expression levels to observed phenotypes

  • Use Western blotting with quantitative standards to normalize protein levels across experiments

• Genetic Complementation:

  • Perform rescue experiments with wild-type protein in knockout backgrounds

  • Introduce point mutations in functional domains to create separation-of-function alleles

  • Use heterologous expression systems to validate phenotypes in different genetic backgrounds

• Membrane Integrity Verification:

  • Monitor membrane potential using voltage-sensitive dyes

  • Measure non-specific leakage with fluorescent tracers of different sizes

  • Assess lipid organization using fluorescent lipid probes

• Control Protein Implementation:

  • Include structurally similar but functionally distinct membrane proteins as controls

  • Use scrambled or reverse-sequence protein variants as negative controls

  • Implement temperature-sensitive alleles to enable temporal control of protein function

• Statistical Validation:

  • Apply appropriate statistical tests for multiple comparisons

  • Implement blinded analysis protocols for subjective measurements

  • Use quasi-experimental research designs when randomization is not possible

By systematically addressing these potential sources of artifacts, researchers can more confidently attribute observed effects to USA300HOU_2320 function rather than experimental variables or non-specific membrane perturbations.

What computational approaches can predict functional domains in USA300HOU_2320 to guide experimental design?

Computational approaches offer valuable insights for predicting functional domains within USA300HOU_2320, enabling more targeted experimental designs:

• Transmembrane Topology Prediction:

  • Apply multiple prediction algorithms (TMHMM, Phobius, HMMTOP) and develop consensus models

  • Validate predictions through site-directed reporter fusion experiments

  • Create topological maps identifying potential functional loops and domains

• Evolutionary Analysis:

  • Perform multiple sequence alignment across diverse bacterial species

  • Identify conserved residues through evolutionary trace analysis

  • Calculate selection pressure (dN/dS ratios) across the protein to identify functionally constrained regions

• Structural Modeling:

  • Generate homology models based on structurally characterized membrane proteins

  • Perform molecular dynamics simulations in explicit membrane environments

  • Identify potential ligand binding pockets using computational docking

• Functional Domain Prediction:

  • Search for known functional motifs using pattern recognition algorithms

  • Implement machine learning approaches trained on characterized membrane proteins

  • Apply covariation analysis to identify co-evolving residue networks

• Integration with Experimental Data:

  • Incorporate accessibility data from cysteine scanning or limited proteolysis

  • Validate predictions through site-directed mutagenesis of key residues

  • Develop testable hypotheses for domain functions based on computational predictions

These computational approaches should be integrated to develop a comprehensive functional map of USA300HOU_2320, prioritizing regions for experimental characterization and guiding the design of targeted functional assays.

What experimental design approaches yield the most reliable results when studying USA300HOU_2320 in antibiotic resistance contexts?

To generate reliable results when investigating USA300HOU_2320 in antibiotic resistance contexts, researchers should implement these methodological best practices:

• Experimental Design Selection:

  • Implement true experimental research designs with randomized group assignments when possible

  • Use Solomon four-group designs to control for testing effects in pre/post-treatment scenarios

  • Develop quasi-experimental approaches when randomization isn't feasible due to biological constraints

• Control Implementation:

  • Include isogenic control strains differing only in USA300HOU_2320 status

  • Implement vector-only controls for recombinant expression studies

  • Use wild-type complementation to confirm phenotype restoration

• Environmental Standardization:

  • Standardize growth conditions (media composition, temperature, growth phase)

  • Control for oxygen availability, which can affect membrane protein expression

  • Maintain consistent antibiotic preparation and storage protocols

• Measurement Standardization:

  • Use standardized antibiotic susceptibility testing methods (CLSI or EUCAST guidelines)

  • Implement blinded assessment for subjective measurements

  • Include internal standards for quantitative assays

• Statistical Analysis Framework:

  • Determine appropriate sample sizes through power analysis

  • Apply suitable statistical tests based on data distribution

  • Control for multiple comparisons when screening multiple antibiotics

• Replication Structure:

  • Include both biological and technical replicates

  • Verify key findings across different S. aureus strain backgrounds

  • Validate results using complementary methodological approaches

This comprehensive approach aligns with the principles of true experimental research design while acknowledging the specific challenges of studying membrane proteins in antibiotic resistance contexts .

How should researchers integrate USA300HOU_2320 studies with broader investigations of MRSA pathogenicity?

Integrating USA300HOU_2320 research into broader MRSA pathogenicity studies requires a systematic approach:

• Contextual Expression Analysis:

  • Compare USA300HOU_2320 expression across diverse clinical isolates with varying virulence profiles

  • Analyze expression during different infection stages (adherence, invasion, persistence)

  • Correlate expression patterns with specific virulence phenotypes

• Genetic Interaction Mapping:

  • Perform double-knockout experiments with known virulence factors

  • Implement transposon-sequencing (Tn-seq) in USA300HOU_2320 mutant backgrounds

  • Identify synthetic phenotypes that emerge only in specific genetic contexts

• Host-Pathogen Interaction Studies:

  • Evaluate USA300HOU_2320 mutant phenotypes in cellular infection models

  • Analyze effects on host immune response activation

  • Assess contributions to intracellular survival within phagocytic cells

• Multi-omics Integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Develop network models incorporating USA300HOU_2320 functions

  • Identify regulatory relationships between USA300HOU_2320 and virulence gene expression

• Translational Applications:

  • Evaluate USA300HOU_2320 as a potential biomarker for virulent MRSA strains

  • Assess its potential as a therapeutic target in combination with conventional antibiotics

  • Investigate evolutionary patterns in clinical isolates with varying USA300HOU_2320 sequences

This integrated approach recognizes that MRSA pathogenicity involves complex adaptive mechanisms , with membrane proteins potentially playing roles in multiple virulence-related processes beyond simple antibiotic resistance.

What are the critical quality control parameters for ensuring reproducible results with recombinant USA300HOU_2320?

Ensuring reproducible results with recombinant USA300HOU_2320 requires stringent quality control at multiple experimental stages:

Researchers should:

• Document all quality control results in a standardized format

• Implement a minimum acceptable criteria checklist before proceeding to experiments

• Store reference samples from each production batch for comparison

• Validate protein stability under experimental conditions before use

• Include positive control proteins of known quality in experimental workflows

These quality control measures ensure that observed experimental outcomes can be attributed to genuine biological properties of USA300HOU_2320 rather than technical variability in protein preparation .

What are the future research directions for USA300HOU_2320 membrane protein studies?

Future research on USA300HOU_2320 membrane protein should focus on several promising directions:

• Structural Biology Frontiers:

  • Complete high-resolution structure determination using advanced cryo-EM techniques

  • Map conformational dynamics through hydrogen-deuterium exchange mass spectrometry

  • Characterize lipid-protein interactions that influence function

• Systems Biology Integration:

  • Define the complete interactome of USA300HOU_2320 in multiple growth conditions

  • Map its position within membrane protein networks affecting antibiotic resistance

  • Develop predictive models for membrane adaptation under antibiotic pressure

• Translational Applications:

  • Evaluate USA300HOU_2320 as a potential drug target for novel anti-MRSA therapeutics

  • Investigate its utility as a biomarker for specific MRSA lineages

  • Develop antibodies or aptamers targeting exposed epitopes for diagnostic applications

• Evolutionary Perspectives:

  • Characterize sequence variation across diverse S. aureus clinical isolates

  • Investigate horizontal gene transfer patterns involving the genomic region

  • Assess functional conservation across related membrane proteins in other pathogens

• Methodological Advancements:

  • Develop improved expression systems for membrane protein production

  • Implement advanced imaging techniques for visualizing USA300HOU_2320 in native contexts

  • Create biosensor applications based on USA300HOU_2320 functional properties

These research directions will contribute to our understanding of membrane protein biology in pathogenic bacteria while potentially yielding new approaches to address the growing challenge of antibiotic resistance in S. aureus .

How does understanding USA300HOU_2320 contribute to broader knowledge of bacterial membrane proteins?

The study of USA300HOU_2320 contributes significantly to our understanding of bacterial membrane proteins through multiple dimensions:

• Structural Insights: As a member of the UPF0060 family of uncharacterized membrane proteins, structural characterization of USA300HOU_2320 would illuminate folding patterns and topological arrangements potentially shared across diverse bacterial species, expanding our fundamental knowledge of membrane protein architecture.

• Functional Paradigms: Elucidating the function of USA300HOU_2320 may reveal novel membrane protein roles in cellular processes, potentially identifying new functional paradigms relevant to other bacterial systems.

• Methodological Advancements: Technical challenges overcome during USA300HOU_2320 studies (expression, purification, reconstitution) can inform approaches for other difficult membrane proteins, contributing to methodological improvements in the field.

• Evolutionary Perspective: Comparative analysis of USA300HOU_2320 homologs across different bacterial species provides insights into membrane protein evolution and adaptation, particularly in the context of antibiotic exposure.

• Pathogen Biology: Understanding USA300HOU_2320's role in S. aureus specifically contributes to our knowledge of how membrane proteins influence pathogenicity, virulence, and antibiotic resistance mechanisms in clinically relevant bacteria .

These contributions extend beyond S. aureus biology, offering broader implications for membrane protein research across diverse bacterial systems and potentially informing therapeutic strategies targeting this important class of proteins.

What is the recommended protocol for expressing and purifying USA300HOU_2320 for structural studies?

The following protocol is recommended for high-yield expression and purification of USA300HOU_2320 for structural studies:

Expression Protocol:

• Bacterial Strain Selection:

  • Use E. coli strain BL21(DE3) or C41(DE3) (specialized for membrane proteins)

  • Transform with pET-based vector containing codon-optimized USA300HOU_2320 with N-terminal His tag

• Culture Conditions:

  • Grow in Terrific Broth supplemented with appropriate antibiotic

  • Incubate at 37°C until OD600 reaches 0.6-0.8

  • Induce with 0.5 mM IPTG

  • Shift temperature to 18°C for 16-18 hours to promote proper folding

• Cell Harvest:

  • Collect cells by centrifugation (5,000 × g, 15 min, 4°C)

  • Wash once with phosphate buffer

  • Store pellet at -80°C or proceed to purification

Purification Protocol:

• Cell Lysis:

  • Resuspend cells in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, protease inhibitors)

  • Disrupt by sonication or high-pressure homogenization

  • Add detergent (recommended: 1% DDM) and stir for 1 hour at 4°C

• Initial Purification:

  • Clear lysate by centrifugation (100,000 × g, 1 hour, 4°C)

  • Apply supernatant to Ni-NTA column equilibrated with buffer containing 0.05% DDM

  • Wash with 20-50 mM imidazole

  • Elute with 250-300 mM imidazole gradient

• Secondary Purification:

  • Perform size exclusion chromatography using Superdex 200 column

  • Use running buffer containing 0.05% DDM, 150 mM NaCl, 20 mM Tris-HCl pH 8.0

• Quality Assessment:

  • Verify purity by SDS-PAGE (should be >90%)

  • Confirm identity by mass spectrometry

  • Assess monodispersity by dynamic light scattering

• Storage:

  • Concentrate to 5-10 mg/mL using 30 kDa cutoff concentrators

  • Add glycerol to 20% final concentration

  • Flash-freeze in liquid nitrogen and store at -80°C