Recombinant Staphylococcus aureus UPF0754 membrane protein SaurJH1_1933 (SaurJH1_1933)

What is UPF0754 membrane protein SaurJH1_1933 and what organism does it originate from?

SaurJH1_1933 is a membrane protein from Staphylococcus aureus strain JH1, classified as belonging to the UPF0754 protein family. The protein consists of 374 amino acids and is encoded by the SaurJH1_1933 gene . S. aureus is a major human pathogen responsible for both community and hospital-acquired infections, with particular clinical significance due to the evolution of methicillin-resistant strains (MRSA) that demonstrate intrinsic resistance to a wide range of β-lactam antibiotics .

The full-length protein is characterized by its membrane association, suggested by its amino acid composition that includes hydrophobic regions typical of transmembrane domains. The protein's isolation in detergent-resistant membrane (DRM) fractions of S. aureus cells indicates its localization within specific membrane microdomains .

What expression system is used for recombinant SaurJH1_1933 production and what tags are incorporated?

Recombinant SaurJH1_1933 is produced using an in vitro Escherichia coli expression system . The recombinant protein is typically engineered with an N-terminal 10xHis-tag to facilitate purification through affinity chromatography and detection in experimental applications .

What are the optimal storage conditions for recombinant SaurJH1_1933?

For optimal stability and activity retention, recombinant SaurJH1_1933 should be stored according to the following guidelines:

• Store at -20°C for routine use, or at -80°C for extended storage

• Avoid repeated freeze-thaw cycles, which can significantly reduce protein activity

• For lyophilized format: store at -20°C/-80°C with a typical shelf life of 12 months

• For liquid format: store at -20°C/-80°C with a typical shelf life of 6 months

• Working aliquots can be stored at 4°C for up to one week

The protein is typically supplied in a Tris/PBS-based buffer (pH 8.0) containing 6% trehalose for lyophilized preparations, or with 50% glycerol for liquid preparations to enhance stability during freezing .

How should recombinant SaurJH1_1933 be reconstituted for experimental use?

Proper reconstitution of SaurJH1_1933 is critical for maintaining protein activity. Follow these methodological steps:

• If lyophilized, allow the protein vial to reach room temperature before opening

• Reconstitute using a Tris-based buffer at pH 8.0, which is optimized for this protein's stability

• Gently mix by inversion or mild vortexing until completely dissolved

• Avoid introducing bubbles during reconstitution

• For membrane proteins like SaurJH1_1933 with potential hydrophobic regions, consider adding a mild detergent (e.g., 0.1% Triton X-100) to aid solubilization if recommended by the manufacturer

• Aliquot immediately after reconstitution to avoid repeated freeze-thaw cycles

• Store reconstituted aliquots according to recommended storage conditions

Some hydrophobic proteins may have high isoelectric points causing the molecule to be hydrophobic at neutral pH. In such cases, acidic reconstitution buffers might be necessary to ensure complete solubilization and prevent protein loss through adsorption to labware surfaces .

What is the significance of carrier-free versus BSA-containing preparations of SaurJH1_1933?

The designation "CF" (Carrier-Free) in product codes like CSB-CF413418SUI indicates the absence of carrier proteins such as bovine serum albumin (BSA) . The choice between carrier-free and BSA-containing preparations has significant methodological implications:

Researchers should select the appropriate format based on their specific experimental requirements. For studies requiring protein quantification, immunoprecipitation, or protein labeling, carrier-free preparations are preferable to avoid interference from BSA .

How can SaurJH1_1933 be studied in the context of bacterial membrane microdomains?

Membrane proteins like SaurJH1_1933 often localize to specific membrane microdomains, which can be critical for their function. Advanced methodological approaches for studying SaurJH1_1933 in membrane microdomains include:

• Membrane fractionation: Isolate detergent-resistant membrane (DRM) fractions using non-ionic detergents and density gradient centrifugation. Compare protein distribution between DRM and detergent-soluble membrane (DSM) fractions using immunoblotting with anti-His tag antibodies .

• Super-resolution microscopy: Employ stimulated emission depletion microscopy (STED) using FLAG-tagged SaurJH1_1933 variants and fluorescently labeled antibodies to visualize membrane distribution patterns with nanometer resolution .

• Protein-protein interaction studies: Use co-immunoprecipitation or pull-down assays with His-tagged SaurJH1_1933 to identify interacting membrane proteins, particularly scaffold proteins like flotillin (FloA) that may facilitate its oligomerization and function .

• Split-protein reassembly approaches: Create fusion constructs with split fluorescent proteins (e.g., YFP) to visualize protein-protein interactions in situ, which can reveal the spatial organization of SaurJH1_1933 within the membrane .

These methodologies can provide critical insights into how SaurJH1_1933 associates with other membrane components and potentially contributes to S. aureus pathogenicity.

What potential roles might SaurJH1_1933 play in S. aureus virulence and antibiotic resistance?

While specific functional data on SaurJH1_1933 is limited in the provided search results, its membrane localization in S. aureus suggests potential roles in virulence and antibiotic resistance that warrant investigation:

• Membrane integrity and stress response: As a membrane protein, SaurJH1_1933 may contribute to cell envelope integrity or stress responses that influence antibiotic susceptibility, particularly in MRSA strains with mortality rates of approximately 20% in clinical settings .

• Protein complex scaffold interactions: Given that flotillin scaffold proteins in S. aureus facilitate assembly of multiprotein complexes involved in virulence, SaurJH1_1933 may participate in similar scaffold-dependent complexes that regulate pathogenicity .

• RNA metabolism connections: If SaurJH1_1933 interacts with membrane-associated RNA processing machinery (like the degradosome containing RNase Rny), it could influence expression of virulence factors through modulation of regulatory sRNAs such as rsaA and sau63 .

• Potential drug target: Small molecules that modulate the oligomerization or function of membrane proteins have shown promise in reducing S. aureus virulence potential both in vitro and in vivo, suggesting similar approaches might be valuable if SaurJH1_1933's role in pathogenicity is established .

Future research directions should include phenotypic characterization of SaurJH1_1933 deletion mutants and investigation of its potential interactions with known virulence-associated protein complexes in S. aureus.

What analytical techniques are recommended for studying SaurJH1_1933 structure and oligomerization?

To elucidate the structure and potential oligomerization states of SaurJH1_1933, researchers should consider the following analytical approaches:

• Circular Dichroism (CD) Spectroscopy: Analyze secondary structure composition of purified recombinant SaurJH1_1933, particularly to assess helical content expected in transmembrane domains.

• Size Exclusion Chromatography (SEC): Evaluate oligomeric states under various detergent conditions to determine if SaurJH1_1933 forms monomers, dimers, or higher-order oligomers.

• Blue Native PAGE: Separate native protein complexes containing SaurJH1_1933 to identify potential interaction partners and complex sizes.

• Crosslinking Studies: Apply chemical crosslinkers of varying spacer arm lengths to capture transient protein-protein interactions involving SaurJH1_1933.

• FRET Analysis: Create fluorescently labeled SaurJH1_1933 constructs to measure protein proximity and dynamics in membrane environments.

• Microscale Thermophoresis (MST): Quantify interactions between SaurJH1_1933 and potential binding partners with high sensitivity in solution.

• Antibody-free Protein Detection: Utilize in-gel protein detection methods to avoid interference from S. aureus non-specific immunoglobulin-binding proteins that often generate false positives in immunoblotting experiments .

These analytical approaches should be complemented with computational methods such as homology modeling and molecular dynamics simulations to predict structural features based on the amino acid sequence.

How can recombinant SaurJH1_1933 be incorporated into S. aureus functional studies?

When designing functional studies of SaurJH1_1933 in S. aureus, researchers should consider the following methodological approaches:

• Chromosomal Integration: Generate S. aureus strains with chromosomally integrated tagged versions of SaurJH1_1933 (e.g., SaurJH1_1933-FLAG) expressed under the control of its native promoter to maintain physiological expression levels .

• Deletion Mutant Construction: Create precise gene deletion mutants (ΔSaurJH1_1933) using allelic replacement techniques to evaluate phenotypic changes related to virulence, stress response, and antibiotic susceptibility.

• Complementation Studies: Reintroduce SaurJH1_1933 using plasmid-based expression systems to verify phenotypes observed in deletion mutants are specifically due to loss of this protein.

• Subcellular Localization: Employ cell fractionation followed by immunodetection to determine the precise membrane localization of SaurJH1_1933, particularly its distribution between detergent-resistant and detergent-soluble membrane fractions .

• Protein-Protein Interaction Analysis: Use pull-down assays with His-tagged recombinant SaurJH1_1933 to identify interaction partners from S. aureus lysates, with verification through reciprocal co-immunoprecipitation .

• In vivo Infection Models: Evaluate the contribution of SaurJH1_1933 to S. aureus pathogenicity using appropriate infection models such as murine or invertebrate systems .

What considerations should be made when using recombinant SaurJH1_1933 for antibody production and immunological studies?

When using recombinant SaurJH1_1933 for antibody production and immunological studies, researchers should consider these important methodological factors:

• Antigen Preparation: Use carrier-free preparations of recombinant SaurJH1_1933 to avoid generating antibodies against carrier proteins like BSA that could complicate subsequent analyses .

• Epitope Accessibility: Consider that membrane proteins like SaurJH1_1933 have hydrophobic regions that may be poorly immunogenic or inaccessible in the native protein conformation.

• Cross-Reactivity Testing: Thoroughly test antibody specificity against recombinant protein, whole cell lysates, and membrane fractions of both wild-type and ΔSaurJH1_1933 mutant strains.

• Protein A Interference: Be aware that S. aureus produces Protein A, which non-specifically binds immunoglobulins and can generate false positives in immunoblotting. Consider using antibody-free protein detection methods for verification .

• Adjuvant Selection: Choose adjuvants carefully when immunizing animals, as inappropriate adjuvants may denature membrane proteins and generate antibodies that fail to recognize the native conformation.

• Immunization Schedule: Implement longer immunization protocols with multiple booster injections to overcome the typically lower immunogenicity of membrane proteins.

• Application-Specific Validation: Validate antibodies separately for each application (Western blot, immunoprecipitation, immunofluorescence) as performance can vary significantly between techniques.

These considerations are crucial for generating reliable immunological reagents for studying SaurJH1_1933 function in S. aureus.

How can solubility and stability issues with recombinant SaurJH1_1933 be addressed?

Membrane proteins like SaurJH1_1933 often present solubility and stability challenges. The following methodological approaches can help overcome these issues:

• Buffer Optimization: Test various buffer compositions, particularly focusing on:

  • pH range (7.0-8.5)

  • Salt concentration (100-500 mM)

  • Addition of glycerol (5-20%)

  • Inclusion of trehalose (6%) as a stabilizing agent

• Detergent Selection: Screen multiple detergents for optimal solubilization:

  • Mild non-ionic detergents (e.g., DDM, CHAPS)

  • Zwitterionic detergents (e.g., LDAO)

  • Determine critical micelle concentration (CMC) for each detergent

• Temperature Considerations: Maintain protein at 4°C during purification and handling procedures to minimize degradation.

• Aggregation Prevention: Add reducing agents (e.g., DTT or β-mercaptoethanol) to prevent disulfide-mediated aggregation if cysteine residues are present.

• Aliquoting Strategy: Prepare single-use aliquots immediately after reconstitution to avoid repeated freeze-thaw cycles that significantly reduce protein activity .

• Storage Vessel Selection: Use low-protein binding tubes for storage to prevent loss through adsorption to container surfaces .

By systematically addressing these factors, researchers can significantly improve the handling and experimental utility of recombinant SaurJH1_1933.