Recombinant Escherichia fergusonii Protein AaeX (aaeX)

What is the AaeX protein in Escherichia fergusonii?

AaeX is a protein found in Escherichia fergusonii (strain ATCC 35469 / DSM 13698 / CDC 0568-73) encoded by the aaeX gene. This protein is identified in the gene database as EFER_3220. The commercially available recombinant versions are typically produced as partial sequences rather than the full-length protein. When designing experiments, researchers should consider whether the partial nature of the recombinant protein includes functional domains relevant to their research questions. Protein sequence analysis and comparison with homologous proteins in related species can help identify conserved domains and potential functions .

What is known about the physiological function of AaeX in E. fergusonii?

Based on current research, the physiological function of AaeX in E. fergusonii has not been completely characterized. Researchers investigating this protein should consider comparative genomics approaches by analyzing functions of AaeX homologs in related species such as E. coli. Experimental approaches to determine function might include gene knockout studies followed by phenotypic analysis, protein-protein interaction studies, and structural analysis to identify potential binding sites or catalytic domains. Researchers might also explore whether AaeX contributes to pathogenicity or antimicrobial resistance, as E. fergusonii has been identified as an emerging pathogen with resistance mechanisms such as mobile colistin resistance genes .

How stable is recombinant AaeX protein under laboratory conditions?

The stability of recombinant AaeX protein depends significantly on storage and handling conditions. The commercially available lyophilized powder formulations offer excellent stability during shipping and long-term storage. After reconstitution, the protein should be maintained at a concentration of 0.1-1.0 mg/mL in deionized sterile water. For optimal stability during extended storage, adding glycerol to a final concentration of 5-50% is recommended, followed by aliquoting and storing at -20°C/-80°C. Repeated freeze-thaw cycles should be avoided as they can lead to protein degradation and loss of activity. Researchers should perform stability testing specific to their experimental conditions, particularly if planning assays that require extended incubation periods .

What expression systems are available for recombinant AaeX production?

Multiple expression systems have been validated for recombinant AaeX production:

The choice of expression system should be guided by specific experimental requirements. For structural studies requiring native conformation, insect or mammalian expression systems might be preferable despite higher costs. For applications where post-translational modifications are not critical, the E. coli system offers economic advantages .

What purification methods are most effective for recombinant AaeX?

The optimal purification strategy for recombinant AaeX depends on the expression system and the tags incorporated during expression. For His-tagged variants, immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins provides high specificity and yield. For biotinylated versions produced using the Avi-tag system, streptavidin affinity chromatography offers exceptional purity. In both cases, researchers should consider including a secondary purification step such as size exclusion chromatography to remove aggregates and ensure homogeneity. Purification protocols should be optimized for buffer conditions (pH, salt concentration) that maintain protein stability and activity. Quality control should include SDS-PAGE analysis to confirm purity (>85% as specified for commercial products) and mass spectrometry to verify protein identity .

How can I optimize the biotinylation of AaeX using the Avi-tag system?

The Avi-tag biotinylation system provides site-specific labeling of recombinant AaeX through the action of E. coli biotin ligase (BirA). This enzyme catalyzes the formation of an amide linkage between biotin and a specific lysine residue within the 15-amino acid AviTag sequence. For optimal biotinylation:

• Ensure sufficient biotin in the culture medium (typically 50 μM)

• Co-express the BirA ligase or add purified enzyme

• Maintain optimal temperature (30°C) during the biotinylation reaction

• Verify biotinylation efficiency using streptavidin shift assays or mass spectrometry

The biotinylated AaeX (product code CSB-EP487580EOR1-B) offers advantages for applications requiring protein immobilization, such as surface plasmon resonance, pull-down assays, or microscopy studies. The extremely high affinity between biotin and streptavidin (Kd ≈ 10^-15 M) ensures stable capture while the site-specific nature of the biotinylation minimizes interference with protein function .

What methods are recommended for analyzing AaeX protein-protein interactions?

To investigate AaeX protein-protein interactions, researchers should consider employing multiple complementary approaches:

• Co-immunoprecipitation (Co-IP): Using antibodies against AaeX or its interaction partners to pull down protein complexes

• Yeast two-hybrid screening: For systematic identification of potential binding partners

• Surface plasmon resonance (SPR): For quantitative measurement of binding kinetics and affinity

• Crosslinking mass spectrometry: To identify interaction interfaces at amino acid resolution

• Biolayer interferometry: For real-time, label-free detection of molecular interactions

The biotinylated version of recombinant AaeX (CSB-EP487580EOR1-B) is particularly valuable for SPR and pull-down assays due to the site-specific attachment of biotin. When designing these experiments, researchers should consider controls to distinguish specific from non-specific interactions and validate identified interactions through multiple independent methods .

How can I assess the structural characteristics of recombinant AaeX?

Several techniques can be employed to characterize the structural properties of recombinant AaeX:

• Circular dichroism (CD) spectroscopy: To analyze secondary structure content (α-helices, β-sheets)

• Thermal shift assays: To evaluate protein stability and identify stabilizing buffer conditions

• Size exclusion chromatography with multi-angle light scattering (SEC-MALS): To determine oligomeric state and homogeneity

• Nuclear magnetic resonance (NMR) spectroscopy: For detailed structural analysis of smaller proteins or domains

• X-ray crystallography: For high-resolution structural determination

For accurate structural analysis, researchers should ensure high purity (>95%) and homogeneity of the protein sample. The choice between E. coli, yeast, baculovirus, or mammalian expression systems may significantly impact structural characteristics, especially if post-translational modifications influence folding. Comparative analysis across different expression systems might provide insights into structural variations and their functional implications .

What computational approaches can complement experimental studies of AaeX?

Computational methods can provide valuable insights to guide experimental research on AaeX:

• Homology modeling: Generating structural models based on related proteins with known structures

• Molecular dynamics simulations: Investigating conformational dynamics and potential binding sites

• Protein-protein docking: Predicting interaction interfaces with potential binding partners

• Functional annotation through conserved domains: Identifying potential functional sites based on sequence conservation

Recent advancements in structural prediction tools like AlphaFold2 can provide high-confidence structural models even in the absence of closely related template structures. These computational predictions can guide the design of experimental studies, such as site-directed mutagenesis to validate functional residues. The integration of computational and experimental approaches often yields more comprehensive understanding than either approach alone .

How can AaeX be utilized in vaccine development against E. fergusonii?

Recombinant AaeX may serve as a candidate antigen for vaccine development against E. fergusonii infections. Key considerations for this application include:

• Epitope mapping: Identifying B-cell and T-cell epitopes within AaeX that can elicit protective immune responses

• Antigenicity assessment: Evaluating the ability of AaeX or its epitopes to stimulate immune recognition

• Adjuvant selection: Determining optimal adjuvants to enhance immune responses to AaeX

• Delivery systems: Developing appropriate formulations and delivery mechanisms

Recent research has employed immunoinformatics approaches to design multi-epitope vaccines against E. fergusonii by analyzing surface-exposed virulent proteins. While research specifically on AaeX in this context is limited, similar methodologies could be applied. If AaeX is confirmed as a virulence factor or surface-exposed protein, its epitopes might be incorporated into multi-epitope vaccine constructs. Molecular docking studies with immune receptors such as TLR-4, MHC-I, and MHC-II could predict immunogenic potential, followed by molecular dynamics simulations to assess stability of the interactions .

What role might AaeX play in antimicrobial resistance mechanisms?

While AaeX has not been directly implicated in antimicrobial resistance based on the provided search results, E. fergusonii strains have been found to harbor mobile colistin resistance (mcr) genes. Researchers investigating AaeX should consider potential associations with resistance mechanisms through:

• Comparative expression analysis: Examining AaeX expression levels in resistant versus susceptible strains

• Gene knockout studies: Assessing whether AaeX deletion affects antimicrobial susceptibility profiles

• Protein-protein interaction studies: Identifying potential interactions between AaeX and known resistance mediators

• Structural analysis: Determining if AaeX shares structural features with proteins involved in resistance

Research has identified E. fergusonii strains carrying mcr-1 genes on different plasmid types (IncX4, IncI2, and IncHI2), conferring resistance to colistin. Additionally, chromosomal mutations, particularly in two-component regulatory systems like PhoP/PhoQ, contribute to resistance phenotypes. Understanding whether AaeX interacts with these resistance mechanisms could provide insights into potential therapeutic interventions .

How can recombinant AaeX be used to study E. fergusonii pathogenesis?

Recombinant AaeX can serve as a valuable tool for investigating E. fergusonii pathogenesis through several approaches:

• Functional characterization: Determining whether AaeX contributes to virulence through in vitro and in vivo models

• Host-pathogen interaction studies: Assessing AaeX interactions with host cell components

• Immune response analysis: Evaluating host immune recognition and response to AaeX

• Comparative studies: Analyzing differences in AaeX sequence, structure, or expression between clinical and environmental isolates

If AaeX is identified as a virulence factor, the recombinant protein could be used to develop inhibitors or neutralizing antibodies as potential therapeutic agents. The availability of recombinant AaeX with different tags and from different expression systems provides flexibility for diverse experimental approaches, from structural studies to functional assays .

What are common challenges in working with recombinant AaeX and how can they be addressed?

Researchers may encounter several challenges when working with recombinant AaeX:

For troubleshooting specific experimental issues, researchers should systematically vary conditions and maintain detailed records of protocol modifications and outcomes. Consulting literature on similar proteins from related bacterial species may provide additional insights into optimizing work with AaeX .

How should I design controls for experiments involving recombinant AaeX?

Appropriate controls are essential for experiments involving recombinant AaeX:

• Negative controls:

  • Buffer-only conditions to assess background signals

  • Irrelevant proteins of similar size/properties to evaluate specificity

  • Heat-denatured AaeX to distinguish activity-dependent effects

• Positive controls:

  • Well-characterized proteins with similar functions, if known

  • Validated interaction partners, if studying protein-protein interactions

  • Known substrates or ligands, if enzymatic or binding activities are being assessed

• Validation controls:

  • Multiple detection methods to confirm observations

  • Concentration gradients to establish dose-dependence

  • Time-course experiments to characterize kinetics

The specific controls will depend on the experimental design and research questions. For example, when using biotinylated AaeX in pull-down assays, controls should include non-biotinylated protein and streptavidin beads without protein to identify non-specific binding .

How can I validate antibodies for detecting native AaeX in E. fergusonii samples?

Validating antibodies for AaeX detection requires systematic testing:

• Specificity validation:

  • Western blot analysis comparing wild-type E. fergusonii with aaeX knockout strains

  • Competition assays with purified recombinant AaeX

  • Testing cross-reactivity with lysates from related bacterial species

• Sensitivity assessment:

  • Serial dilutions of recombinant AaeX to establish detection limits

  • Comparison of different antibody clones or sources

  • Optimization of detection methods (colorimetric, chemiluminescent, fluorescent)

• Application-specific validation:

  • For immunofluorescence: verify localization patterns match predictions

  • For immunoprecipitation: confirm enrichment of AaeX and known partners

  • For ELISA: establish standard curves with recombinant protein

Researchers should be aware that antibody performance may vary between applications (Western blot vs. immunoprecipitation vs. immunofluorescence). The partial recombinant AaeX proteins available commercially might generate antibodies that recognize only specific domains, potentially limiting detection of certain forms of the native protein .

How does AaeX from E. fergusonii compare to homologous proteins in other bacterial species?

Understanding the evolutionary relationships and functional conservation of AaeX requires comparative analysis:

• Sequence comparison: Perform multiple sequence alignment of AaeX homologs across Enterobacteriaceae to identify conserved domains and species-specific variations

• Phylogenetic analysis: Construct phylogenetic trees to understand evolutionary relationships

• Structural comparison: Compare predicted or determined structures to identify conserved structural features

• Functional conservation: Assess whether homologs in different species share similar functions or contexts

Researchers investigating E. fergusonii have conducted core genome analysis using tools like Roary to identify core genes and build phylogenetic trees. Similar approaches could be applied specifically to AaeX and its homologs. The close relationship between E. fergusonii and E. coli suggests potential functional similarities, but species-specific adaptations may exist and should be considered when extrapolating findings between species .

What is known about AaeX expression patterns in different E. fergusonii strains?

Expression patterns of AaeX may vary between different E. fergusonii strains and under different conditions:

• Strain variation: Compare expression levels across clinical isolates, environmental strains, and reference strains

• Condition-dependent expression: Assess expression under various growth conditions, stresses, or host environments

• Regulatory mechanisms: Identify transcription factors and regulatory elements controlling aaeX expression

• Co-expression networks: Determine whether aaeX is co-regulated with other genes as part of specific pathways

Phylogenetic analysis has shown that E. fergusonii strains exhibit diverse relationships. For example, strains carrying mcr-1 genes have been found to cluster separately from other strains, suggesting potential differences in gene expression patterns or regulatory networks. Researchers should consider these strain-specific variations when designing experiments and interpreting results related to AaeX expression and function .