Recombinant Shigella boydii serotype 4 Protein AaeX (aaeX)

What is Recombinant Shigella boydii serotype 4 Protein AaeX and what are its structural characteristics?

Recombinant Shigella boydii serotype 4 Protein AaeX (aaeX) is a full-length protein derived from Shigella boydii serotype 4 (strain Sb227). The protein has the UniProt identifier Q31WA9 and contains 67 amino acids in its expression region (1-67). The complete amino acid sequence is: MSLFPVIVVFGLSFPPIFFELLLSLAIFWLVRRVLVPTGIYDFVWHPALFNTALYCCLFYLISRLFV . The protein is part of the gene locus SBO_3147 and represents the full-length protein as found in the native organism . Within the broader context of Shigella research, AaeX belongs to a genus of Gram-negative, nonspore-forming, nonmotile, facultative aerobic, rod-shaped bacteria that causes disease primarily in primates including humans .

What are the optimal storage conditions for maintaining stability of Recombinant Shigella boydii serotype 4 Protein AaeX?

The stability of Recombinant Shigella boydii serotype 4 Protein AaeX is contingent upon proper storage conditions. For routine laboratory use, the protein should be stored at -20°C in its storage buffer (Tris-based buffer with 50% glycerol, optimized specifically for this protein) . For extended storage periods, conservation at either -20°C or -80°C is recommended. To preserve protein integrity, repeated freeze-thaw cycles should be strictly avoided as they can lead to protein denaturation and functional degradation . A best practice approach involves creating working aliquots upon initial thawing, which can be stored at 4°C for up to one week to minimize freeze-thaw damage while maintaining experimental consistency . This aliquoting strategy should be incorporated into experimental design to ensure reproducibility across extended research timelines.

How does the functional characterization of AaeX protein differ between recombinant forms and native expression in Shigella boydii?

The functional characterization of recombinant versus natively-expressed AaeX protein requires careful experimental design to account for potential differences in post-translational modifications, protein folding, and functional activity. While recombinant AaeX provides a controlled protein source with defined expression regions (amino acids 1-67) , native expression within Shigella boydii may involve interactions with other bacterial components that influence protein function.

Experimental approaches to characterize these differences should include: (1) comparative enzymatic activity assays between recombinant and native forms; (2) structural analysis using circular dichroism or X-ray crystallography to identify folding variations; and (3) interaction studies with potential binding partners using pull-down assays or surface plasmon resonance. When analyzing protein function within the broader context of Shigella pathogenesis, researchers should consider that Shigella species are classified by three serogroups and one serotype (S. dysenteriae, S. flexneri, S. boydii, and S. sonnei), with S. flexneri being most frequently isolated worldwide (approximately 60% of cases) .

What are the recommended protocols for using Recombinant Shigella boydii serotype 4 Protein AaeX in ELISA-based detection systems?

When implementing Recombinant Shigella boydii serotype 4 Protein AaeX in ELISA-based detection systems, researchers should follow a systematic approach that accounts for the protein's specific characteristics. Begin by determining optimal coating concentration through titration experiments (typically 1-10 μg/ml) in carbonate-bicarbonate buffer (pH 9.6) . The standard quantity available (50 μg) should be carefully allocated across experiments, with consideration given to creating a standard curve using known concentrations.

For reproducible results, consider the following protocol elements:

• Coat microplate wells with optimized concentration of AaeX protein (2-5 μg/ml) and incubate overnight at 4°C

• Block with 1-3% BSA in PBS for 1-2 hours at room temperature

• Apply primary antibodies against AaeX or test samples at appropriate dilutions

• Detect using enzyme-conjugated secondary antibodies and appropriate substrate

When validating results, compare detection limits and specificity against other serotypes of Shigella to establish assay parameters. Given that the tag type for the recombinant protein is determined during the production process , researchers should verify whether the tag might interfere with epitope recognition in immunological assays.

How can Recombinant Shigella boydii serotype 4 Protein AaeX be effectively employed in antimicrobial resistance studies?

Utilizing Recombinant Shigella boydii serotype 4 Protein AaeX in antimicrobial resistance (AMR) studies requires integration with established microbiological methods. The Enterics for Global Health (EFGH) Shigella surveillance study methodology provides a valuable framework . Researchers should consider a dual approach:

First, for protein-drug interaction studies:

• Perform binding assays between purified AaeX protein and antimicrobial compounds

• Analyze structural changes using circular dichroism spectroscopy upon drug binding

• Assess functional changes in protein activity in the presence of antimicrobials

Second, for contextualizing results within broader AMR patterns:

• Compare findings with antimicrobial susceptibility testing (AST) results from clinical isolates

• Employ standardized methods like the Kirby-Bauer disc diffusion following Clinical and Laboratory Standards Institute (CLSI) guidelines

• Test against relevant antibiotics including ampicillin, azithromycin, ceftriaxone, ciprofloxacin, and trimethoprim-sulfamethoxazole

The growing emergence of highly resistant Shigella strains necessitates precise characterization of AMR profiles . AaeX-focused studies should be integrated with whole genome sequencing data to establish relationships between protein expression patterns and resistance phenotypes, providing critical information for future Shigella vaccine development strategies.

What methods are most effective for analyzing AaeX protein interactions with host cell receptors?

For analyzing AaeX protein interactions with host cell receptors, researchers should implement a multi-modal approach that combines in vitro binding studies with functional assays. The following methodological framework is recommended:

• Binding Kinetics Analysis:

  • Surface plasmon resonance (SPR) using recombinant AaeX (50 μg quantity as typically supplied) immobilized on sensor chips

  • Isothermal titration calorimetry (ITC) to determine thermodynamic parameters of binding

  • Fluorescence polarization assays for high-throughput screening of potential interaction partners

• Cellular Localization Studies:

  • Confocal microscopy using fluorescently-labeled AaeX protein to track interaction with host cells

  • Subcellular fractionation followed by western blotting to identify compartmentalization

• Functional Validation:

  • Cell-based reporter assays measuring downstream signaling activation

  • Competition assays with known Shigella virulence factors

When interpreting results, consider that Shigella causes disease specifically in primates but not other mammals , suggesting species-specific host receptor interactions. Data analysis should account for the potential influence of the protein tag (determined during production) on binding characteristics by including appropriate controls with different tag configurations.

How can comparative proteomics be applied to understand the evolutionary significance of AaeX across different Shigella serotypes?

Applying comparative proteomics to understand AaeX evolution across Shigella serotypes requires a systematic analysis framework that integrates both computational and experimental approaches. Researchers should begin with sequence alignment of AaeX homologs from different Shigella serotypes, including S. boydii serotype 4 (Q31WA9) , S. flexneri serotype 5b , and other serotypes to identify conserved domains and variable regions.

For comprehensive evolutionary analysis, implement the following methodology:

• Phylogenetic Analysis:

  • Construct maximum likelihood trees based on AaeX sequence alignments

  • Calculate evolutionary distances using appropriate substitution models

  • Map protein variations to known functional domains

• Structural Comparison:

  • Model protein structures using homology modeling based on the known sequence (MSLFPVIVVFGLSFPPIFFELLLSLAIFWLVRRVLVPTGIYDFVWHPALFNTALYCCLFYLISRLFV)

  • Compare predicted structural features across serotypes

  • Identify potential structure-function relationships

• Functional Conservation Assessment:

  • Express recombinant AaeX proteins from multiple serotypes

  • Compare biochemical activities through standardized assays

  • Correlate sequence variations with functional differences

This approach should be interpreted within the broader context of Shigella evolution, considering that S. flexneri represents approximately 60% of worldwide isolates . Results should inform understanding of selective pressures on AaeX conservation or divergence across the genus.

What bioinformatic approaches are most effective for predicting potential epitopes in Recombinant Shigella boydii serotype 4 Protein AaeX for vaccine development?

Effective epitope prediction for Recombinant Shigella boydii serotype 4 Protein AaeX in vaccine development requires an integrated bioinformatic approach that combines sequence-based predictions with structural analysis. Based on the known 67-amino acid sequence (MSLFPVIVVFGLSFPPIFFELLLSLAIFWLVRRVLVPTGIYDFVWHPALFNTALYCCLFYLISRLFV) , researchers should employ the following methodological framework:

• Sequential Epitope Prediction:

  • Apply multiple prediction algorithms (IEDB, BepiPred, ABCpred) to identify B-cell linear epitopes

  • Use machine learning approaches that incorporate physicochemical properties

  • Validate predictions across algorithms and identify consensus epitopes

• Structural Epitope Mapping:

  • Generate 3D structural models of AaeX using homology modeling

  • Identify surface-exposed regions with high accessibility

  • Calculate electrostatic potential to identify charged patches likely to interact with antibodies

• Population Coverage Analysis:

  • Predict MHC-I and MHC-II binding for identified epitopes

  • Analyze epitope conservation across Shigella strains

  • Estimate population coverage based on HLA allele frequencies

The increasing emergence of antimicrobial-resistant Shigella strains highlights the importance of vaccine development approaches. Researchers should consider that preservation of isolates allows for unequivocal confirmation by whole genome sequencing , which can inform epitope selection by identifying conserved regions under low selective pressure.

How does AaeX protein expression correlate with antimicrobial resistance patterns in clinical Shigella boydii isolates?

Investigating the correlation between AaeX protein expression and antimicrobial resistance (AMR) patterns in clinical Shigella boydii isolates requires a comprehensive approach that integrates proteomics with microbiological methods. Researchers should design studies that:

• Quantify AaeX Expression Levels:

  • Develop quantitative Western blot protocols using antibodies against recombinant AaeX

  • Implement targeted proteomics using mass spectrometry with isotope-labeled standards

  • Measure aaeX gene expression using RT-qPCR in parallel with protein quantification

• Determine AMR Profiles:

  • Perform antimicrobial susceptibility testing following CLSI guidelines as described in the EFGH Shigella surveillance methodology

  • Test against relevant antibiotics including ampicillin, azithromycin, ceftriaxone, ciprofloxacin, nalidixic acid, mecillinam, and trimethoprim-sulfamethoxazole

  • Determine minimum inhibitory concentrations (MICs) for ambiguous results

• Correlation Analysis:

  • Apply multivariate statistical methods to identify associations between AaeX expression levels and specific resistance patterns

  • Control for confounding variables such as geographical origin and patient demographics

  • Validate findings across multiple clinical isolates

The preservation of isolates from surveillance studies allows for additional investigations into microbial ecology, virulence factors, and AMR determinants . Researchers should contextualize their findings within the growing concern about extensively drug-resistant (XDR) Shigella strains, which show resistance to multiple antibiotics including ampicillin, ciprofloxacin, trimethoprim-sulfamethoxazole, third-generation cephalosporins, and azithromycin .

What are the critical quality control parameters for ensuring experimental reproducibility when working with Recombinant Shigella boydii serotype 4 Protein AaeX?

Ensuring experimental reproducibility with Recombinant Shigella boydii serotype 4 Protein AaeX requires rigorous quality control measures across the experimental workflow. Critical parameters include:

• Protein Integrity Verification:

  • SDS-PAGE analysis to confirm molecular weight and purity

  • Western blotting using tag-specific or AaeX-specific antibodies

  • Mass spectrometry to verify sequence integrity, particularly focusing on the full-length sequence (MSLFPVIVVFGLSFPPIFFELLLSLAIFWLVRRVLVPTGIYDFVWHPALFNTALYCCLFYLISRLFV)

• Storage and Handling Protocol:

  • Strict adherence to recommended storage conditions (-20°C or -80°C for extended storage)

  • Implementation of an aliquoting system to avoid freeze-thaw cycles

  • Maintenance of working aliquots at 4°C for no more than one week

• Functional Activity Assessment:

  • Establishment of standardized activity assays specific to AaeX

  • Inclusion of positive and negative controls in each experimental batch

  • Regular benchmarking against reference standards

• Documentation and Reporting:

  • Detailed recording of protein lot numbers, production dates, and handling history

  • Comprehensive reporting of buffer compositions and experimental conditions

  • Implementation of a laboratory information management system (LIMS) to track sample history

Researchers should also consider that the tag type for the recombinant protein is determined during the production process , which necessitates consistent sourcing or additional controls when changing suppliers to ensure comparable results across studies.

How can researchers effectively differentiate between the biological activities of AaeX and other Shigella proteins in experimental systems?

Differentiating the biological activities of AaeX from other Shigella proteins requires a systematic approach that combines molecular, biochemical, and functional techniques. Researchers should implement the following methodological framework:

• Molecular Specificity Controls:

  • Use gene knockout or CRISPR-Cas9 edited Shigella strains lacking aaeX

  • Complement with recombinant AaeX to confirm phenotype restoration

  • Employ siRNA or antisense oligonucleotides for targeted suppression in expression systems

• Biochemical Discrimination:

  • Develop AaeX-specific antibodies using unique epitopes based on the amino acid sequence

  • Perform immunoprecipitation to isolate AaeX-specific protein complexes

  • Use affinity purification followed by mass spectrometry to identify specific interaction partners

• Functional Differentiation:

  • Design domain-swapping experiments between AaeX and related proteins

  • Conduct structure-function analysis through systematic mutagenesis

  • Develop reporter systems that selectively respond to AaeX activity

When interpreting results, consider the broader context of Shigella taxonomy and characteristics. Shigella is closely related to E. coli and represents one of the leading bacterial causes of diarrhea worldwide . The genus includes four main species: S. dysenteriae, S. flexneri, S. boydii, and S. sonnei , each with distinct protein profiles that may show functional overlap with AaeX.

What are the key considerations for scaling up production of Recombinant Shigella boydii serotype 4 Protein AaeX for research purposes?

Scaling up production of Recombinant Shigella boydii serotype 4 Protein AaeX for research purposes requires careful optimization of expression systems, purification protocols, and quality control measures. While avoiding commercial production questions, researchers should consider these methodological aspects:

• Expression System Optimization:

  • Evaluate multiple expression hosts (E. coli, yeast, baculovirus, or mammalian cells) similar to approaches used for related Shigella proteins

  • Optimize codon usage for the expression host

  • Test different promoters and induction conditions for maximum yield while maintaining protein integrity

  • Consider the impact of the tag type, which is determined during the production process

• Purification Strategy Development:

  • Design a multi-step purification protocol combining affinity chromatography with size exclusion or ion exchange methods

  • Optimize buffer composition based on the known storage buffer (Tris-based buffer with 50% glycerol)

  • Implement quality control checkpoints at each purification stage

• Stability Enhancement:

  • Formulate storage conditions to maximize stability (-20°C or -80°C for long-term storage)

  • Investigate lyophilization or spray-drying techniques for room temperature stability

  • Test different additives to prevent aggregation and maintain activity

• Quality Assessment Framework:

  • Develop analytical methods to confirm protein identity, purity, and activity

  • Establish acceptance criteria for batch-to-batch consistency

  • Implement stability testing protocols for different storage conditions

The standard quantity of commercially available protein (50 μg) may be insufficient for extensive research, necessitating in-house scale-up while maintaining the full expression region (amino acids 1-67) and functional integrity.

What are the most promising research directions for understanding AaeX's role in Shigella pathogenesis?

Understanding AaeX's role in Shigella pathogenesis presents several promising research directions that integrate molecular, cellular, and in vivo approaches. Based on current knowledge of AaeX from Shigella boydii serotype 4 and the broader context of Shigella research , investigators should consider:

• Host-Pathogen Interaction Studies:

  • Identify potential host cell receptors that interact with AaeX

  • Characterize the role of AaeX in adhesion, invasion, or intracellular survival

  • Investigate differential expression of AaeX during various stages of infection

• Structural Biology Approaches:

  • Determine the three-dimensional structure of AaeX using X-ray crystallography or cryo-EM

  • Map functional domains within the 67-amino acid sequence

  • Compare structural features with homologous proteins in related pathogens

• Systems Biology Integration:

  • Analyze AaeX within the context of the Shigella virulome

  • Perform global interaction studies to place AaeX in pathogenesis networks

  • Develop mathematical models predicting AaeX contribution to virulence

• Translational Applications:

  • Evaluate AaeX as a diagnostic biomarker for Shigella infections

  • Assess AaeX as a potential vaccine candidate

  • Investigate AaeX as a target for novel antimicrobial development

These research directions should be contextualized within the growing concern about antimicrobial resistance in Shigella . The preservation of isolates allows for additional studies on microbial ecology, virulence factors, AMR determinants, and epidemiology across regions and globally , providing valuable resources for AaeX-focused research.

How might advances in structural biology techniques enhance our understanding of AaeX function and potential applications?

Advances in structural biology techniques offer unprecedented opportunities to deepen our understanding of AaeX function and expand its potential applications. Based on the known amino acid sequence of Recombinant Shigella boydii serotype 4 Protein AaeX (MSLFPVIVVFGLSFPPIFFELLLSLAIFWLVRRVLVPTGIYDFVWHPALFNTALYCCLFYLISRLFV) , researchers should leverage:

• High-Resolution Structure Determination:

  • Cryo-electron microscopy for visualization of AaeX in different conformational states

  • NMR spectroscopy to analyze dynamic regions and binding interfaces

  • X-ray crystallography to obtain atomic-level details of protein structure

• Molecular Dynamics Simulations:

  • Simulate AaeX behavior in membrane environments

  • Model conformational changes upon interaction with potential binding partners

  • Predict functional effects of point mutations within the 67-amino acid sequence

• Integrative Structural Biology:

  • Combine multiple techniques (small-angle X-ray scattering, hydrogen-deuterium exchange mass spectrometry) to build comprehensive structural models

  • Apply cross-linking mass spectrometry to identify interaction surfaces

  • Use computational approaches to predict functional sites

The integration of structural data with antimicrobial resistance studies is particularly relevant given the growing concern about extensively drug-resistant Shigella strains . Structural insights can guide the development of novel therapeutic approaches and inform vaccine design strategies to address the challenges posed by antimicrobial resistance in Shigella infections.

What collaborative research frameworks would best advance our understanding of AaeX in the context of global Shigella surveillance?

Advancing our understanding of AaeX within global Shigella surveillance requires multi-disciplinary collaborative frameworks that integrate laboratory research with epidemiological monitoring. Based on approaches in the Enterics for Global Health (EFGH) Shigella surveillance study , an effective framework should include:

• Integrated Surveillance Network:

  • Establish standardized protocols for AaeX detection and characterization across surveillance sites

  • Implement common microbiological methods for Shigella isolation following established procedures

  • Develop centralized biorepositories for strain collection and distribution

• Multi-Omics Approach:

  • Coordinate genomics, transcriptomics, and proteomics analyses across research groups

  • Integrate AaeX expression data with antimicrobial resistance profiles

  • Link phenotypic characteristics to genetic determinants

• Data Sharing Infrastructure:

  • Create open-access databases for AaeX sequence variants

  • Develop visualization tools for geographic distribution of variants

  • Implement machine learning algorithms to identify emerging patterns

• Translational Pipeline:

  • Establish collaborations between academic institutions, public health agencies, and vaccine developers

  • Design community-based studies to assess the impact of AaeX variation on disease outcomes

  • Develop rapid diagnostic tools targeting AaeX for field deployment