Recombinant Salmonella heidelberg Protein AaeX (aaeX)

What are the fundamental characteristics of Recombinant Salmonella heidelberg Protein AaeX?

While the search results don't provide specific information about AaeX, recombinant Salmonella heidelberg proteins are typically expressed in E. coli expression systems with affinity tags (such as His-tags) to facilitate purification. Based on related Salmonella heidelberg proteins, these recombinant proteins are commonly produced as full-length constructs and purified to high homogeneity (>90% as determined by SDS-PAGE) . For experimental work, researchers should consider that recombinant versions may contain fusion tags that could potentially influence protein folding or activity compared to native versions.

What expression systems yield optimal results for Salmonella heidelberg protein production?

E. coli is the predominant expression system for Salmonella heidelberg proteins, as evidenced by production methods for other recombinant proteins from this organism . When expressing membrane or secreted proteins like AaeX, optimization of induction conditions (temperature, IPTG concentration, and induction duration) becomes critical for proper folding and solubility. Expression in E. coli BL21(DE3) or similar strains with reduced protease activity is typically recommended, particularly for full-length constructs (comparable to the 1-719 amino acid construct described for Aas protein) .

How do storage conditions affect the stability of recombinant Salmonella heidelberg proteins?

Recombinant Salmonella heidelberg proteins require careful storage to maintain activity. Based on protocols for similar proteins, storage at -20°C/-80°C is recommended, with aliquoting necessary to avoid repeated freeze-thaw cycles . The proteins are typically supplied in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0, which helps maintain stability during freeze-thaw processes . For working stocks, storage at 4°C is suitable for up to one week, but longer-term storage necessitates the addition of glycerol (5-50% final concentration) and storage at -80°C .

What reconstitution protocols are recommended for lyophilized Salmonella heidelberg proteins?

For optimal reconstitution of lyophilized Salmonella heidelberg proteins:

• Centrifuge the vial briefly before opening to bring contents to the bottom

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

• Add glycerol to a final concentration of 5-50% (with 50% being standard practice)

• Aliquot for long-term storage at -20°C/-80°C

This protocol minimizes protein degradation and maintains activity across experiments. Avoid repeated freeze-thaw cycles as they significantly reduce protein stability and activity. When planning experiments, account for approximately 10-15% loss of activity after each freeze-thaw cycle.

What analytical methods are most effective for characterizing Salmonella heidelberg protein structure and function?

For comprehensive characterization of Salmonella heidelberg proteins, a multi-method approach is recommended:

SDS-PAGE remains the standard method for initial purity assessment, with expected purity exceeding 90% for research-grade recombinant proteins . For membrane proteins like AaeX, detergent selection during purification and analysis significantly impacts structural integrity.

How should researchers design experiments to study protein-protein interactions involving Salmonella heidelberg proteins?

When designing experiments to study protein-protein interactions:

• Begin with in silico prediction of potential interaction partners based on pathway analysis

• Perform pull-down assays using the His-tagged recombinant protein as bait

• Validate interactions through reciprocal co-immunoprecipitation

• Quantify binding affinity using surface plasmon resonance or isothermal titration calorimetry

• Confirm biological relevance using in vivo techniques (FRET, BiFC)

For Salmonella heidelberg proteins, consider their native environment and physiological context. Membrane proteins like AaeX may require specialized conditions including appropriate detergents or lipid reconstitution to maintain native conformation during interaction studies. When planning experiments, account for how the His-tag might affect interactions and consider including tag removal via protease cleavage .

How can recombinant Salmonella heidelberg proteins be utilized to study antimicrobial resistance mechanisms?

Recombinant Salmonella heidelberg proteins serve as valuable tools for investigating antimicrobial resistance (AMR) mechanisms. S. Heidelberg has demonstrated concerning levels of resistance, particularly to ceftiofur/ceftriaxone, requiring focused research on resistance determinants .

Research approaches should include:

• Structure-function studies of proteins implicated in resistance (efflux pumps, β-lactamases)

• Protein-antibiotic binding assays to determine interaction mechanisms

• Mutagenesis studies to identify critical residues involved in resistance

• In vitro reconstitution of enzymatic activities (e.g., β-lactamase activity)

• Crystallography or cryo-EM to determine protein structures with bound antibiotics

Notably, S. Heidelberg isolates have shown a propensity to acquire and disseminate multiple plasmids encoding for multidrug resistance, and whole genome sequencing has demonstrated close relationships between blaCMY-2 containing plasmids . This suggests horizontal plasmid dissemination rather than just clonal spread of particular strains, making recombinant protein studies particularly valuable for understanding resistance mechanisms.

What role do Salmonella heidelberg proteins play in virulence and pathogenicity?

Salmonella heidelberg proteins contribute significantly to virulence through multiple mechanisms that can be investigated using recombinant proteins:

• Adhesion to host cells (fimbrial proteins)

• Invasion of epithelial cells (Type III secretion system components)

• Intracellular survival (stress response proteins)

• Toxin production (enterotoxins)

• Immune evasion (surface-modifying enzymes)

At the molecular level, virulence genes located on chromosomes and plasmids, including those contained in Salmonella pathogenicity islands, have been identified in S. Heidelberg . These genes encode factors that facilitate endothelial uptake, as well as regulatory and effector virulence factors for adhesion, invasion, and toxin production . S. Heidelberg isolates carry a variety of phages, virulence genes (including 62 pathogenicity and 13 fimbrial markers), and/or IncX plasmids that contribute to colonization and persistence .

The co-presence of AMR and virulence genes is particularly concerning, as studies have demonstrated that S. Heidelberg causes damage to intestinal mucosa similar to that caused by Salmonella enterica subsp. enterica serovar Enteritidis .

How can structural biology approaches enhance our understanding of Salmonella heidelberg protein function?

Structural biology approaches provide crucial insights into protein function through detailed molecular architecture analysis:

When applying these techniques to Salmonella heidelberg proteins, researchers should account for the often challenging nature of membrane protein crystallization. For AaeX or similar proteins, detergent screening is critical, with common detergents including DDM, LMNG, or amphipols maintaining native-like environments. Molecular dynamics simulations can complement experimental structures to elucidate conformational changes relevant to function.

How should researchers interpret contradictory results when studying Salmonella heidelberg protein interactions?

When encountering contradictory results in protein interaction studies:

• Verify protein quality through multiple analytical methods (SDS-PAGE, mass spectrometry)

• Assess the impact of different experimental conditions (pH, salt concentration, temperature)

• Compare results across multiple interaction detection methods (pull-down, SPR, ITC)

• Evaluate the influence of protein tags on interaction behavior

• Consider the role of post-translational modifications in modulating interactions

For Salmonella proteins specifically, remember that their function may be context-dependent. For example, S. Heidelberg proteins involved in antimicrobial resistance could display different behaviors depending on the presence of specific antibiotics or stress conditions . The environmental conditions (such as temperature, pH) used in experiments should mimic those encountered by the bacterium during infection or colonization.

What bioinformatic approaches are most valuable for analyzing Salmonella heidelberg protein function?

Comprehensive bioinformatic analysis should include:

• Sequence alignment and phylogenetic analysis to identify evolutionary relationships

• Domain prediction to identify functional modules within the protein

• Secondary structure prediction to inform experimental design

• Homology modeling based on related structures

• Molecular docking to predict ligand interactions

• Prediction of post-translational modifications

For Salmonella heidelberg proteins, comparative genomic approaches are particularly valuable given the availability of multiple sequenced strains. The identification of mobile genetic elements containing integrons and clusters of resistance and/or virulence genes has been crucial for understanding pathogenicity . Whole genome sequencing data can inform protein function prediction through genomic context analysis and identification of co-evolved proteins.

What are the most common challenges in expressing Salmonella heidelberg membrane proteins and how can they be addressed?

Common challenges in membrane protein expression include:

For Salmonella heidelberg membrane proteins specifically, screening multiple detergents is critical for maintaining native-like structures. Additionally, expression as fusion proteins with solubility-enhancing tags may improve yields, though tag removal may be necessary for functional studies .

What quality control measures should be implemented when working with recombinant Salmonella heidelberg proteins?

A robust quality control pipeline should include:

• Purity assessment by SDS-PAGE (≥90% purity standard)

• Identity confirmation by mass spectrometry and/or Western blotting

• Homogeneity evaluation by size exclusion chromatography

• Activity assessment through appropriate functional assays

• Stability monitoring over time and storage conditions

• Endotoxin testing if intended for immunological studies

Documentation should include batch records tracking expression conditions, purification steps, and quality metrics. For Salmonella heidelberg proteins, thermal stability assays (such as differential scanning fluorimetry) can provide valuable information about protein folding and buffer optimization. Regular testing of aliquots stored under different conditions can help establish reliable shelf-life expectations and optimal storage protocols .