Recombinant Staphylococcus aureus UPF0060 membrane protein SA2130 (SA2130)

What is UPF0060 membrane protein SA2130 and what organism does it originate from?

UPF0060 membrane protein SA2130 is a membrane-associated protein derived from Staphylococcus aureus strain N315. It is classified in the UPF0060 protein family, which consists of proteins with unknown functions that require further characterization. The protein is encoded by the SA2130 gene locus in the S. aureus genome. This protein is of particular interest because it is derived from S. aureus, a Gram-positive, round-shaped bacterium that belongs to the Firmicutes phylum and is commonly found in the upper respiratory tract and on human skin .

What is the amino acid sequence and structural characteristics of SA2130?

The full amino acid sequence of SA2130 is: mLYPIFIFILAGLCEIGGGYLIWLWLREGQSSLVGLIGGAILmLYGVIATFQSFPSFGRVYAAYGGVFIIMSLIFAMVVDKQMPDKYDVIGAIICIVGVLVmLLPSRA . The protein spans amino acids 1-108 of the native sequence and represents the full-length protein. The sequence contains predominantly hydrophobic amino acids, which is consistent with its classification as a membrane protein. The presence of multiple hydrophobic regions suggests multiple transmembrane domains, which is a common characteristic of integral membrane proteins. The protein has been assigned the UniProt accession number P67149, which can be used to access additional structural and functional information .

How is the recombinant SA2130 protein typically stored and what are the recommended handling conditions?

Recombinant SA2130 protein is typically supplied in a Tris-based buffer containing 50% glycerol, specifically optimized for this protein's stability . For storage, it is recommended to keep the protein at -20°C for regular use, or at -80°C for extended storage periods to maintain optimal activity and structural integrity . For active research, working aliquots can be stored at 4°C for up to one week to minimize freeze-thaw cycles . It is important to note that repeated freezing and thawing is not recommended as it can lead to protein denaturation and loss of activity . When handling the protein, researchers should consider using low-protein binding tubes and working quickly on ice to minimize degradation during experimental procedures.

What purification strategies are most effective for isolating SA2130 with high purity and yield?

Purification of membrane proteins like SA2130 presents unique challenges due to their hydrophobic nature and tendency to aggregate. An effective purification strategy typically begins with membrane fraction isolation followed by solubilization using appropriate detergents. For affinity purification, the recombinant protein is often produced with an affinity tag, though tag selection depends on the expression system and downstream applications . Common purification techniques include immobilized metal affinity chromatography (IMAC) for His-tagged proteins, followed by size exclusion chromatography to remove aggregates and achieve higher purity. For membrane proteins specifically, detergent exchange during purification may be necessary to maintain protein stability and functionality. The final purification protocol should be optimized based on the specific properties of SA2130 and the intended experimental applications.

How can researchers verify the structural integrity and functionality of purified recombinant SA2130?

Verifying the structural integrity and functionality of purified recombinant SA2130 requires multiple analytical approaches. Initially, SDS-PAGE and Western blotting can confirm protein size and identity, while mass spectrometry provides precise molecular weight determination and can verify sequence integrity. For structural assessment, circular dichroism spectroscopy helps evaluate secondary structure elements, particularly important for membrane proteins like SA2130 where proper folding is crucial. Size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) can determine oligomeric state and homogeneity. For functional verification, researchers should develop specific assays based on hypothesized protein functions, potentially including lipid binding assays, proteoliposome reconstitution, or interaction studies with known binding partners. Since SA2130 is a membrane protein, reconstitution into artificial membrane systems may be necessary to assess its native-like behavior and function in a lipid environment.

What are the known or predicted functions of SA2130 in Staphylococcus aureus?

The UPF0060 membrane protein SA2130 belongs to a family of proteins with unknown function (UPF), indicating that its precise biological role remains to be fully elucidated. Based on its membrane localization and sequence characteristics, it is predicted to function in membrane transport, signaling, or structural organization within the bacterial cell membrane. Its presence in the clinically significant S. aureus strain N315 suggests potential involvement in pathogenesis, antibiotic resistance, or bacterial survival mechanisms. Comparative genomic analyses with other bacterial species containing UPF0060 family proteins may provide insights into conserved functions. Understanding SA2130's function is particularly important given that S. aureus can act as both a commensal organism and an opportunistic pathogen, causing a wide range of infections from minor skin conditions to life-threatening diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome, bacteremia, and sepsis .

How can SA2130 be utilized in Staphylococcus aureus detection systems?

Recombinant SA2130 protein can serve as a valuable tool in developing specific detection systems for S. aureus. It can be used as a positive control in immunoassays or as an antigen for generating specific antibodies that target S. aureus. In phage-based detection systems, understanding protein interactions between bacterial membrane proteins and bacteriophages is crucial. Research on phage-based detection of S. aureus has shown that bacterial proteins can significantly influence detection efficiency . While the search results don't specifically mention SA2130 in phage-based detection, the methodologies described for S. aureus detection could potentially be applied or modified for systems involving SA2130. For instance, researchers could investigate whether SA2130 interacts with protein A or other surface proteins that affect phage-based detection methods. Additionally, recombinant SA2130 could potentially be incorporated into ELISA-based detection systems for specific identification of S. aureus strains expressing this protein.

What role might SA2130 play in Staphylococcus aureus pathogenicity and virulence?

While specific evidence directly linking SA2130 to S. aureus pathogenicity is limited in the provided search results, its nature as a membrane protein suggests potential roles in host-pathogen interactions, biofilm formation, or environmental adaptation. Membrane proteins in pathogenic bacteria often contribute to virulence through multiple mechanisms, including adhesion to host tissues, evasion of host immune responses, nutrient acquisition, and antibiotic resistance. S. aureus is known to produce various virulence factors, including potent protein toxins and cell-surface proteins that bind and inactivate antibodies . As a membrane protein, SA2130 could be involved in maintaining membrane integrity under stress conditions encountered during infection. To investigate this potential role, researchers might conduct comparative studies between wild-type S. aureus and SA2130 knockout mutants, examining differences in virulence in appropriate infection models. Additionally, interaction studies with host proteins or antimicrobial compounds could reveal whether SA2130 contributes to pathogenicity or antibiotic resistance mechanisms.

What are the challenges in detecting Staphylococcus aureus in serum samples and how can they be overcome?

Detection of S. aureus in serum samples presents significant challenges due to the inhibitory effects of serum components on detection methods. Research has shown that human serum potently inhibits phage-based detection of S. aureus, with IgG identified as a causative agent in this inhibition . The inhibitory activity has been observed with both whole IgG and its Fc and F(ab')2 fragments . This inhibition is not due to reduced bacterial growth or loss of phage viability but rather appears to be related to interference with phage-host interactions.

A notable solution to overcome this challenge is the use of protein A, which has been demonstrated to neutralize the inhibitory effects of serum on S. aureus detection . A modified phage cocktail containing recombinant protein A has shown promise broadly across different S. aureus strains, eliminating the need for additional purification steps or extended enrichment periods . This approach significantly improves the detection sensitivity in serum-containing matrices, allowing for reliable detection of approximately 100 CFU of S. aureus strains that would otherwise be below the detection threshold in the presence of serum .

How can recombinant SA2130 be incorporated into experimental designs for studying membrane protein function?

Incorporating recombinant SA2130 into experimental designs for studying membrane protein function requires strategic approaches that preserve the protein's native structure and activity. Researchers can utilize artificial membrane systems such as liposomes, nanodiscs, or lipid bilayers to reconstitute SA2130 in a native-like environment. This reconstitution allows for functional studies including ion conductance, substrate transport, or interaction with other membrane components.

For structural studies, recombinant SA2130 can be prepared for techniques such as X-ray crystallography, cryo-electron microscopy, or nuclear magnetic resonance spectroscopy, though membrane proteins present unique challenges for these methods. Site-directed mutagenesis of specific amino acid residues in the SA2130 sequence can help identify functionally important regions and delineate structure-function relationships. Additionally, fluorescently labeled SA2130 can be used in localization studies to determine its distribution within bacterial cells or artificial membrane systems.

Interaction studies using techniques such as pull-down assays, surface plasmon resonance, or crosslinking followed by mass spectrometry can identify potential binding partners and help elucidate SA2130's function within the S. aureus membrane. These experimental approaches should be customized based on specific research questions about SA2130's role in bacterial physiology or pathogenesis.

What controls and validation steps are necessary when working with recombinant SA2130 in research applications?

When working with recombinant SA2130, implementing appropriate controls and validation steps is crucial to ensure experimental reliability and reproducibility. Initially, protein identity and purity should be confirmed through SDS-PAGE, Western blotting, and mass spectrometry analysis. For functional assays, researchers should include both positive and negative controls specific to the experimental design. For instance, in binding studies, a known interacting protein serves as a positive control, while a non-related protein provides a negative control.

Testing multiple protein concentrations and establishing dose-response relationships helps validate functional observations. When recombinant SA2130 is used in detection methods, calibration curves with known quantities of the purified protein should be established, and the limit of detection and quantification should be determined . For researchers investigating SA2130's role in S. aureus biology, complementation experiments comparing wild-type, knockout, and complemented strains provide strong validation of observed phenotypes.

To ensure batch-to-batch consistency, quality control measures should include activity assays and stability testing under various storage conditions. When reporting results, researchers should provide detailed information about the recombinant protein's expression system, purification method, buffer composition, and storage conditions to facilitate reproduction by other laboratories .

How do strain variations in SA2130 sequence and expression impact experimental outcomes?

Strain variations in SA2130 sequence and expression levels can significantly impact experimental outcomes and interpretations. The SA2130 protein characterized in most studies is derived from S. aureus strain N315, but sequence variations may exist across different clinical and laboratory strains . These variations might include single nucleotide polymorphisms, insertions/deletions, or differences in regulatory elements affecting expression levels. Such genetic differences can translate to functional variations that influence bacterial physiology, pathogenicity, or detection assay performance.

When studying SA2130's function or utilizing it in detection systems, researchers should consider sequencing the gene from their specific strains of interest and comparing it to reference sequences. Additionally, expression analysis using quantitative PCR or proteomics approaches can reveal strain-specific differences in SA2130 abundance that might correlate with phenotypic variations. For comprehensive studies, including a panel of diverse S. aureus strains representing different lineages and isolation sources would provide more robust and generalizable results.

What are the current contradictions or knowledge gaps in understanding SA2130's structure-function relationship?

The structure-function relationship of SA2130 remains largely uncharacterized, representing a significant knowledge gap in S. aureus research. As a member of the UPF0060 family (Uncharacterized Protein Family), its biological function has not been definitively established. The amino acid sequence suggests multiple transmembrane domains, but high-resolution structural data is currently lacking. Without crystal or cryo-EM structures, precise information about protein folding, transmembrane arrangement, and potential active sites remains speculative.

Additionally, while we know SA2130 is a membrane protein, its specific subcellular localization (e.g., cytoplasmic membrane vs. membrane microdomains) and topology (orientation within the membrane) have not been thoroughly investigated. Understanding these aspects is crucial for hypothesizing about potential functions such as transport, signaling, or structural roles. There is also limited information about SA2130's potential interaction partners or whether it functions as a monomer or forms higher-order oligomeric structures.

Current research methodologies for membrane proteins present technical challenges that have likely contributed to these knowledge gaps. Addressing these limitations will require innovative approaches combining genetic, biochemical, and advanced structural biology techniques. Comparative genomics across multiple bacterial species containing UPF0060 family proteins could also provide valuable insights into conserved structural features and potential functional roles.

How can advanced techniques like cryo-EM, molecular dynamics simulations, or CRISPR-Cas9 gene editing advance our understanding of SA2130?

Advanced techniques provide powerful approaches to resolve current knowledge gaps regarding SA2130. Cryo-electron microscopy (cryo-EM) offers advantages for membrane protein structural determination without the need for crystallization, potentially revealing SA2130's three-dimensional architecture, transmembrane domain organization, and structural motifs associated with function. Single-particle cryo-EM or tomography could elucidate whether SA2130 functions as a monomer or forms higher-order complexes.

Molecular dynamics simulations can complement structural studies by modeling SA2130's behavior within lipid bilayers, predicting conformational changes, and identifying potential substrate binding sites or interaction interfaces. These simulations can generate testable hypotheses about structure-function relationships, particularly for regions that might be flexible or disordered in static structural models.

CRISPR-Cas9 gene editing enables precise genetic manipulation of S. aureus to create SA2130 knockout strains, point mutations, or tagged variants for functional studies. By comparing phenotypes of wild-type and mutant strains under various conditions (e.g., antibiotic stress, host cell interaction, biofilm formation), researchers can identify physiological roles of SA2130. CRISPR interference (CRISPRi) approaches allow for conditional knockdown to study essential functions.

Integrative approaches combining these advanced techniques with traditional biochemical and microbiological methods would provide comprehensive insights into SA2130's structure, function, and biological significance in S. aureus physiology and pathogenesis. Collaborative research across disciplines (structural biology, microbiology, bioinformatics) would accelerate progress in understanding this uncharacterized protein.