Recombinant Staphylococcus aureus Probable CtpA-like serine protease (SAS1363)

What is the structural composition of SAS1363?

SAS1363 is a 496-amino acid protein with a transmembrane domain located near the N-terminus, suggesting it may be membrane-associated. The protein contains characteristic serine protease domains, including the catalytic triad essential for its enzymatic activity. According to sequence data, it begins with MDDKQHTSSSDDERAEIATSNQDQETNSSKRVHLKRWQFISILIGTILITAVITVVAYIFINQK and contains multiple functional domains required for its proteolytic activity . The protein is encoded by the SAS1363 gene locus in S. aureus strain MSSA476, as documented in UniProt (Q6G9E1) .

How is SAS1363 classified within bacterial proteases?

SAS1363 is classified as a probable CtpA-like serine protease (EC=3.4.21.-), belonging to the larger family of trypsin-like serine proteases. These enzymes typically cleave peptide bonds using a catalytic triad consisting of serine, histidine, and aspartic acid residues. Bacterial CtpA proteases are often involved in protein maturation processes, including C-terminal processing of various proteins, which can be essential for bacterial viability and virulence .

What are the typical storage and handling conditions for recombinant SAS1363?

Recombinant SAS1363 is typically stored in Tris-based buffer with 50% glycerol to maintain stability. For long-term storage, the protein should be kept at -20°C or -80°C to preserve activity. Working aliquots can be maintained at 4°C for up to one week. Repeated freeze-thaw cycles should be avoided as they may compromise protein integrity and enzymatic activity . When working with the protein, temperature-controlled conditions (usually 4°C) are recommended during experimental procedures to minimize degradation.

What is the proposed role of SAS1363 in S. aureus pathogenicity?

While specific information about SAS1363's role in pathogenicity is limited in the provided research, serine proteases in S. aureus generally participate in multiple virulence mechanisms. As a probable CtpA-like protease, SAS1363 may be involved in protein maturation processes essential for bacterial survival and/or virulence factor activation. Bacterial serine proteases are known to degrade host extracellular matrix components, modulate immune responses, and facilitate nutrient acquisition during infection . The membrane-associated nature of SAS1363 suggests it may interact with host cells during infection, potentially contributing to adhesion or invasion processes.

What challenges exist in developing inhibitors against SAS1363?

Developing specific inhibitors against SAS1363 presents several challenges common to protease-targeted drug development. Researchers must address:

• Specificity concerns: Ensuring inhibitors target SAS1363 without affecting human proteases or beneficial microbiota

• Bioavailability issues: Designing inhibitors that can reach the bacterial membrane where SAS1363 is likely located

• Resistance development: Monitoring potential resistance mechanisms that could emerge upon inhibitor application

Studies on other serine protease inhibitors have shown promising results in disease models, including a multi-target serine protease inhibitor (UAMC-00050) that demonstrated efficacy in Dry Eye Disease and Irritable Bowel Syndrome models . Similar approaches might be applicable to SAS1363, though further characterization of its specific enzymatic properties would be necessary.

What expression systems are optimal for producing recombinant SAS1363?

For recombinant production of SAS1363, several expression systems can be considered:

E. coli Expression System:

• Advantages: Fast growth, high protein yields, well-established protocols

• Challenges: Potential improper folding due to the membrane-associated nature of SAS1363

• Optimization: Fusion tags (such as His, GST, or MBP) can improve solubility and facilitate purification

• Considerations: Expression temperature (typically reduced to 16-25°C) and inducer concentration optimization may improve functional protein yield

Alternative Expression Systems:

• Gram-positive hosts (like Bacillus subtilis): May provide better folding environment for a Gram-positive bacterial protein

• Cell-free systems: Useful for potentially toxic proteins

• Mammalian or insect cell systems: For studies requiring proper post-translational modifications

The expression region (amino acids 1-496) encompasses the full-length protein, but researchers should consider whether to include or exclude the transmembrane domain depending on specific experimental needs .

What methods are recommended for assessing SAS1363 enzymatic activity?

Assessment of SAS1363 enzymatic activity requires carefully designed assays:

Substrate Selection:

• Synthetic peptide substrates with chromogenic or fluorogenic leaving groups

• Natural protein substrates identified through proteomic approaches

• Substrate libraries to determine sequence specificity

Activity Assay Parameters:

• Buffer composition: Typically Tris or phosphate buffers (pH 7.0-8.0)

• Temperature: Usually 25-37°C

• Cofactors: Evaluate dependence on divalent cations (Ca²⁺, Mg²⁺)

• Inhibitors: Use class-specific inhibitors (e.g., PMSF, 3,4-dichloroisocoumarin) as controls

Detection Methods:

• Spectrophotometric detection of chromogenic substrate cleavage

• Fluorescence-based assays for increased sensitivity

• Mass spectrometry for natural substrate identification

• Activity-based protein profiling (ABPP) using chemical probes that react with the active enzyme

How can researchers develop activity-based probes for studying SAS1363?

Activity-based protein profiling (ABPP) represents an effective approach for studying SAS1363 in complex biological settings:

Probe Design Considerations:

• Warhead selection: Diaryl phosphonate warheads have shown success with trypsin-like serine proteases

• Recognition element: Include amino acid analogs or basic polar residues as side chains to target the specific binding pocket of SAS1363

• Affinity tag incorporation: "Clickable" tags enable detection, visualization, or affinity purification of labeled enzymes

Probe Validation:

• Test against recombinant SAS1363 to determine labeling efficiency

• Evaluate specificity against other serine proteases

• Assess probe performance in complex biological samples

Kinetic Characterization:

• Determine inhibitory potencies (IC₅₀ values)

• Establish mechanism of inhibition (reversible vs. irreversible)

• Calculate kinetic constants

Recent research has demonstrated that both irreversible and reversible probes can effectively label recombinant proteases. Notably, even reversible slow-tight-binding probes can label proteases under denaturing SDS-PAGE conditions due to the formation of high-affinity complexes and slow dissociation rates .

What is the potential of SAS1363 as a vaccine antigen candidate?

As a surface-associated protease, SAS1363 may represent a potential vaccine antigen candidate against S. aureus infections:

Vaccination Strategies:

• Recombinant protein approach: Using purified SAS1363 or its immunogenic domains

• Multi-antigen formulations: Combining SAS1363 with other S. aureus antigens

• Bioconjugation approach: Conjugating SAS1363 to polysaccharide antigens for enhanced immunogenicity

Considerations for Vaccine Development:

• Antigen conservation across S. aureus strains

• Ability to induce protective antibodies that neutralize enzymatic activity

• Potential cross-reactivity with human proteases

• Adjuvant selection to promote appropriate immune responses

Current S. aureus vaccine research has shown that including multiple antigens and proper adjuvant selection are critical for efficacy. For example, vaccines containing multiple recombinant S. aureus antigens have shown promising results in preclinical studies . The inclusion of SAS1363 in such formulations would require assessment of its immunogenicity and protective potential.

How might SAS1363 contribute to antibiotic resistance mechanisms?

While direct evidence linking SAS1363 to antibiotic resistance is not provided in the search results, bacterial proteases can potentially contribute to resistance mechanisms through several pathways:

Potential Mechanisms:

• Enzymatic degradation of antibiotics containing susceptible peptide bonds

• Processing of cell wall components to reduce antibiotic binding or penetration

• Cleavage of bacterial proteins involved in stress responses to antibiotics

• Modification of bacterial surface structures affecting permeability

Research Approaches:

• Comparing SAS1363 expression levels in resistant versus susceptible strains

• Evaluating effects of SAS1363 knockout/overexpression on antibiotic susceptibility

• Testing SAS1363 inhibitors in combination with antibiotics for synergistic effects

The growing concern over S. aureus antibiotic resistance highlights the importance of understanding the potential role of all virulence factors, including proteases like SAS1363, in resistance mechanisms .

SAS1363 Protein Specifications