Recombinant Staphylococcus aureus UPF0223 protein SAS1032 (SAS1032)

What is Staphylococcus aureus UPF0223 protein SAS1032?

Staphylococcus aureus UPF0223 protein SAS1032 is a recombinant protein from S. aureus that belongs to the UPF0223 protein family. This protein is known by multiple identifiers including SAUSA300_0997 (as designated in the USA300 strain). The UPF0223 designation indicates it belongs to a family of proteins with unknown function that are currently being characterized . The protein has a complete amino acid sequence identified and can be expressed recombinantly in systems such as E. coli for research applications.

What is known about the function of UPF0223 protein?

Currently, the specific biological function of UPF0223 protein in Staphylococcus aureus remains largely uncharacterized. The "UPF" (Uncharacterized Protein Family) designation indicates that while the protein has been identified and sequenced, its precise cellular role and biochemical functions are not yet fully understood. Ongoing research aims to elucidate its function through structural analysis, interaction studies, and comparative genomics approaches across bacterial species.

What expression systems are most effective for recombinant UPF0223 protein?

E. coli is the primary expression system documented for recombinant UPF0223 protein production . This prokaryotic expression system is particularly suitable for bacterial proteins as it provides an environment similar to the native host. When expressing UPF0223 protein in E. coli, researchers should consider:

• Optimizing codon usage for efficient translation

• Selecting appropriate promoter systems for controlled expression

• Using appropriate strains optimized for recombinant protein expression

• Implementing proper induction conditions to maximize yield

The choice of expression system should align with the intended application, as the expression host can influence protein folding, post-translational modifications, and solubility.

What purification methods yield the highest purity for UPF0223 protein?

For optimal purification of recombinant UPF0223 protein, affinity chromatography approaches are recommended based on the tags used during expression. According to available data, the protein can be purified to >85% purity as assessed by SDS-PAGE . The purification strategy should include:

• Initial capture using affinity chromatography based on the fusion tag

• Secondary purification steps such as ion exchange or size exclusion chromatography

• Quality control assessments including SDS-PAGE and potentially mass spectrometry

• Endotoxin removal if the protein is intended for functional or cellular assays

The choice of purification tag should be considered carefully as it may affect the protein's structure and function.

What are the optimal storage conditions for maintaining UPF0223 protein stability?

Optimal storage conditions for recombinant UPF0223 protein depend on the formulation:

• Lyophilized form: Can be stored at -20°C/-80°C with a shelf life of approximately 12 months

• Liquid form: Can be stored at -20°C/-80°C with a shelf life of approximately 6 months

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

The stability of the protein is influenced by several factors including buffer composition, pH, presence of stabilizing agents, and freeze-thaw cycles. It is generally recommended to avoid repeated freeze-thaw cycles as they can lead to protein degradation and loss of activity.

What reconstitution protocols are recommended for lyophilized UPF0223 protein?

For optimal reconstitution of lyophilized UPF0223 protein:

• Briefly centrifuge the vial containing lyophilized protein before opening to ensure all material is at the bottom

• Reconstitute in deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL

• Consider adding glycerol to a final concentration of 5-50% for long-term storage

• Aliquot the reconstituted protein to minimize freeze-thaw cycles

After reconstitution, it is advisable to filter the solution through a 0.22 μm filter to ensure sterility, particularly for applications sensitive to microbial contamination.

Are there structural differences between recombinant and native UPF0223 protein?

• The core structural fold is likely preserved in recombinant expression

• Minor differences might occur in flexible loop regions

• Post-translational modifications present in the native protein may be absent in recombinant forms expressed in E. coli

When assessing structural similarity, methods such as Combinatorial Extension (CE), FATCAT-flexible, and TM-Align are commonly used with metrics including RMSD, TM-score, and analysis of secondary structural elements .

What analytical methods are recommended for assessing UPF0223 protein structure?

To thoroughly characterize the structure of recombinant UPF0223 protein, researchers should consider employing multiple complementary techniques:

• X-ray crystallography for high-resolution structural determination

• NMR spectroscopy for solution structure and dynamics analysis

• Circular dichroism (CD) spectroscopy for secondary structure assessment

• Size exclusion chromatography with multi-angle light scattering (SEC-MALS) for oligomeric state determination

• Differential scanning calorimetry (DSC) or differential scanning fluorimetry (DSF) for thermal stability assessment

When comparing structures obtained through different methods, it's important to note that some differences may be attributed to the methodology rather than actual structural differences in the protein itself .

How is recombinant UPF0223 protein utilized in Staphylococcus research?

Recombinant UPF0223 protein serves multiple research purposes in Staphylococcus aureus studies:

• Structural and functional characterization: Determining the three-dimensional structure and biochemical properties to infer function

• Protein-protein interaction studies: Identifying binding partners within bacterial systems

• Antibody production: Generating specific antibodies for detection and localization studies

• Enzymatic activity assessment: Evaluating potential catalytic functions

• Pathogenicity research: Investigating potential roles in virulence

In clinical microbiology contexts, understanding S. aureus proteins contributes to bacteriuria and other infection studies, though specific applications of UPF0223 in clinical settings require further investigation .

Can UPF0223 protein be used for developing diagnostic tools?

While the specific utility of UPF0223 protein in diagnostic applications is not explicitly documented in the provided literature, recombinant bacterial proteins generally offer potential in developing:

• Serological assays: Detecting antibodies against S. aureus in patient samples

• Immunodiagnostic tools: Creating specific detection systems for S. aureus strains

• Molecular diagnostics: Serving as controls or standards in nucleic acid-based detection systems

Development of such applications would require validation of the protein's specificity, immunogenicity, and correlation with clinical presentations of S. aureus infections.

What quality control parameters should be assessed for recombinant UPF0223 protein?

Comprehensive quality control for recombinant UPF0223 protein should include:

• Purity assessment: SDS-PAGE analysis with a target of >85% purity as indicated in available specifications

• Identity confirmation: Mass spectrometry verification of molecular weight and sequence

• Endotoxin testing: Especially important for applications involving cell culture or immunological studies

• Stability assessment: Evaluating protein integrity after storage under recommended conditions

• Functional testing: Developing appropriate activity assays based on the protein's known or predicted functions

Establishing batch-to-batch consistency through standardized quality control protocols is essential for reproducible research outcomes.

What are the recommended methods for determining UPF0223 protein concentration?

Several methods can be employed to accurately determine UPF0223 protein concentration:

For routine work with purified UPF0223 protein, A280 measurement using the calculated extinction coefficient based on the amino acid sequence is often most practical.

How might post-translational modifications affect UPF0223 protein function?

Post-translational modifications (PTMs) can significantly impact protein function, and their absence in recombinant systems may affect activity. For UPF0223 protein, the following considerations are relevant:

• Phosphorylation sites: Research on other regulatory proteins demonstrates that serine phosphorylation can modulate enzymatic activity, as seen in studies of UBP12 where phosphorylation of serine 327 inhibits protease activity

• E. coli expression limitations: When expressed in E. coli, UPF0223 protein likely lacks PTMs present in native S. aureus, potentially affecting functional studies

• Site-directed mutagenesis approaches: Creating phosphomimetic mutations (e.g., S→D substitutions) or phospho-null mutations (S→A) can help assess the impact of potential phosphorylation sites on protein function

Researchers should consider whether potential PTMs might be relevant to their specific research questions when designing experiments with recombinant UPF0223 protein.

What considerations should be made when designing functional studies with UPF0223?

When designing functional studies with UPF0223 protein, researchers should consider:

• Conserved domains: Analyzing the USP domain within UPF0223 may provide insights into potential functions, similar to how the USP domain in other proteins contains catalytically essential cysteine and histidine residues

• Comparative analysis: Examining homologous proteins across bacterial species to infer function based on evolutionary conservation

• Protein-protein interactions: Employing techniques such as pull-down assays, yeast two-hybrid, or proximity labeling to identify interaction partners

• Phenotypic studies: Assessing the effects of gene knockout or overexpression in S. aureus to understand physiological roles

• Structural analysis: Using the protein's three-dimensional structure to predict potential binding sites or catalytic regions

Since UPF0223 belongs to a family of uncharacterized proteins, combining multiple experimental approaches is recommended to elucidate its biological role.

How do mutations in conserved residues affect UPF0223 protein activity?

While specific mutational studies of UPF0223 are not documented in the provided literature, insights can be drawn from related research on other proteins:

• Mutations in conserved serine residues can significantly alter protein function, as demonstrated in studies of UBP12 where an S327F mutation affected protease activity

• Substitutions within functional domains, particularly those involving changes in amino acid properties (e.g., serine to phenylalanine), can lead to gain-of-function or loss-of-function phenotypes

• When investigating potential UPF0223 mutations, researchers should consider:

  • Conservation of the residue across species

  • Location relative to predicted functional domains

  • Chemical properties of the substituted amino acids

  • Impact on protein stability and folding

Site-directed mutagenesis studies targeting conserved residues would be valuable for elucidating structure-function relationships in UPF0223 protein.