Recombinant Staphylococcus aureus UPF0291 protein SAS1281 (SAS1281)

What is the structural classification of SAS1281 and how does it relate to other S. aureus proteins?

SAS1281 belongs to the UPF0291 protein family found in Staphylococcus aureus. While sharing sequence homology with certain transmembrane protein families, it represents a distinct class with potential functional significance in bacterial growth-related processes. The protein should be considered in the broader context of S. aureus virulence factors, which include well-characterized proteins such as alpha-toxin that contribute significantly to pathogenesis . When investigating SAS1281, researchers should note that S. aureus has a large repertoire of surface components with potential for immunological targeting .

What expression systems are most effective for producing recombinant SAS1281?

For optimal expression of recombinant SAS1281, researchers should consider E. coli-based expression systems with inducible promoters. Similar to other S. aureus recombinant proteins, such as r-ScaF, expression optimization typically involves:

• Selection of an appropriate expression vector (pET or pGEX systems)

• Optimization of induction conditions (IPTG concentration, temperature, duration)

• Evaluation of solubility enhancers (fusion tags, specialized strains)

• Verification of protein identity via Western blotting

When purifying the expressed protein, implementing a multi-step chromatography approach including affinity purification followed by size exclusion chromatography yields the highest purity preparations suitable for downstream applications such as crystallization or immunological studies .

How can researchers verify the functional integrity of recombinant SAS1281?

Functional validation of recombinant SAS1281 requires multiple complementary approaches:

As with other S. aureus proteins studied for vaccine development, it is essential to verify that the recombinant protein maintains immunological properties comparable to the native form .

What post-translational modifications affect SAS1281 function and how can they be characterized?

Recent research has revealed the significance of post-translational modifications (PTMs) in bacterial proteins, particularly in S. aureus. While specific PTMs for SAS1281 require further characterization, researchers should investigate potential lactylation, similar to what has been observed with alpha-toxin .

To characterize PTMs on SAS1281:

• Employ mass spectrometry (LC-MS/MS) to identify modification sites

• Use specific antibodies against protein modifications (e.g., Pan-αKla antibody for lactylated lysines)

• Generate site-directed mutants at potential modification sites

• Compare the functional properties of modified and unmodified forms

Recent discoveries have shown that lactylation at specific lysine residues of S. aureus toxins can significantly alter their cytolytic activity and virulence potential, suggesting a mechanism by which bacteria adapt toxicity to the host environment .

What are the challenges in distinguishing between specific and non-specific effects when evaluating SAS1281 interactions with host factors?

When studying SAS1281 interactions with host factors, researchers must implement rigorous controls to distinguish specific from non-specific effects:

• Use multiple protein controls including:

  • Denatured SAS1281 (heat-treated)

  • Structurally similar but functionally distinct S. aureus proteins

  • Non-relevant proteins of similar size and charge properties

• Implement concentration-dependent binding studies to establish:

  • Saturation kinetics

  • Binding affinity constants (KD)

  • Competition with known ligands

• Validate interactions using orthogonal methods:

  • Pull-down assays

  • Microscale thermophoresis

  • Isothermal titration calorimetry

• Consider the impact of post-translational modifications, as these can significantly alter protein function and interactions, as demonstrated with alpha-toxin lactylation in S. aureus .

What immunization protocols are most effective when evaluating SAS1281 as a potential vaccine candidate?

Based on successful approaches with other S. aureus antigens, researchers should consider the following immunization protocol for SAS1281:

• Preparation of highly purified recombinant protein (>95% purity)

• Formulation with appropriate adjuvants:

  • Freund's adjuvant for preliminary studies

  • Alum-based adjuvants for translational research

  • Novel adjuvants that enhance Th1/Th17 responses

• Immunization schedule:

  • Prime: Day 0

  • Boost: Days 14 and 28

  • Challenge: Day 42

• Immune response assessment:

  • Cellular immunity: IFN-γ and IL-17 production

  • Humoral immunity: Antibody titers with special attention to IgG2a production

  • Protection evaluation: Bacterial burden in organs, survival rates, inflammatory markers

This approach has proven effective for other S. aureus antigens such as ScaF, which demonstrated protective efficacy in murine models of infection .

How can researchers effectively design experiments to evaluate SAS1281 function in the context of S. aureus pathogenesis?

To rigorously evaluate SAS1281's role in pathogenesis, a comprehensive experimental design should include:

• Generation of genetic tools:

  • Gene deletion mutant (ΔSAS1281)

  • Complemented strain

  • Site-directed mutants for key residues

• In vitro functional assays:

  • Growth kinetics in various media

  • Biofilm formation

  • Resistance to antimicrobial peptides

  • Adherence to host cells

• In vivo infection models:

  • Skin infection (abscess model)

  • Systemic infection (sepsis model)

  • Evaluation parameters:

    • Mortality rates

    • Bacterial burden in organs

    • Inflammatory markers in serum and tissues

    • Histopathological examination

• Host response evaluation:

  • Cytokine profiles (IL-6, TNF-α, IL-1β)

  • Neutrophil recruitment and function

  • Adaptive immune responses

This comprehensive approach, similar to that used for studying alpha-toxin and other virulence factors, provides a holistic understanding of protein function in pathogenesis .

What techniques can reliably identify potential protein-protein interactions involving SAS1281 in the bacterial cell?

To identify SAS1281 interaction partners within S. aureus, researchers should employ complementary approaches:

• Affinity-based methods:

  • Co-immunoprecipitation with anti-SAS1281 antibodies

  • Pull-down assays using tagged recombinant SAS1281

  • Chemical cross-linking followed by mass spectrometry

• Genetic methods:

  • Bacterial two-hybrid systems

  • Suppressor mutation analysis

  • Synthetic genetic arrays

• Structural approaches:

  • X-ray crystallography of protein complexes

  • Hydrogen-deuterium exchange mass spectrometry

  • Cryo-electron microscopy

• In silico prediction:

  • Homology-based interaction prediction

  • Protein docking simulations

  • Co-evolution analysis

When interpreting results, researchers should consider that protein function may be regulated by environmental factors such as lactate concentration, as demonstrated for alpha-toxin in S. aureus .

How should researchers address batch-to-batch variability in recombinant SAS1281 preparations?

Ensuring consistent protein quality across preparations requires systematic quality control:

Implementation of standard operating procedures for expression and purification, coupled with thorough documentation of all production parameters, minimizes variability and ensures reproducible experimental outcomes .

What are the optimal storage conditions for maintaining SAS1281 stability for long-term studies?

Based on experience with similar proteins from S. aureus, the following storage recommendations should be considered:

• Short-term storage (1-2 weeks):

  • 4°C in sterile buffer containing protease inhibitors

  • Avoid repeated freeze-thaw cycles

• Medium-term storage (1-6 months):

  • -20°C in buffer containing 10% glycerol

  • Aliquot in single-use volumes

• Long-term storage (>6 months):

  • -80°C in buffer containing 10-20% glycerol

  • Addition of stabilizers such as trehalose or sucrose (5%)

  • Lyophilization for extended storage

• Stability monitoring:

  • Periodic functional assays

  • SE-HPLC to monitor aggregation

  • SDS-PAGE to assess degradation

Researchers should validate these conditions specifically for SAS1281 through stability studies with regular testing intervals.

How does SAS1281 compare with other S. aureus antigens as a potential vaccine candidate?

When evaluating SAS1281 as a vaccine candidate, researchers should consider its properties in comparison to established S. aureus antigens:

• Conservation across clinical isolates:

  • Sequence analysis of SAS1281 across diverse S. aureus lineages

  • Evaluation of expression levels in different strains

  • Assessment of accessibility to immune system

• Immunogenicity profile:

  • Ability to induce robust cellular responses (IFN-γ, IL-17)

  • Balanced antibody response with emphasis on IgG2a

  • Potential for memory formation

• Protection in animal models:

  • Reduction in mortality

  • Decreased bacterial burden in tissues

  • Modulation of inflammatory responses

Similar to the staphylococcal conserved antigen ScaF, which demonstrated protective efficacy in murine models, SAS1281 should be evaluated for its potential as a component of a multivalent prophylactic vaccine .

What combination of experimental techniques would provide the most comprehensive understanding of SAS1281 structure-function relationships?

A multi-technique approach is essential for elucidating SAS1281 structure-function relationships:

This comprehensive approach, similar to studies on alpha-toxin lactylation, would provide insights into how SAS1281 structure relates to its function in S. aureus biology and pathogenesis .