Recombinant Escherichia coli O157:H7 Bifunctional protein aas (aas)
Description
Introduction
The recombinant Escherichia coli O157:H7 bifunctional protein Aas (Aas) is a full-length enzymatic protein encoded by the aas gene (UniProt ID: B5Z4F4). It is produced via recombinant expression in E. coli and purified with an N-terminal His tag for biochemical and functional studies. This protein exhibits dual enzymatic activity, serving as both a 2-acylglycerophosphoethanolamine (2-acyl-GPE) acyltransferase and an acyl-acyl carrier protein (acyl-ACP) synthetase .
Amino Acid Sequence
The full-length sequence includes conserved domains critical for enzymatic activity, including motifs resembling acyl-AMP binding pockets . The sequence is provided in the product specifications .
Bifunctional Enzymatic Activities
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2-Acyl-GPE Acyltransferase
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Acyl-ACP Synthetase
Physiological Significance
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Membrane Homeostasis: Critical for maintaining membrane fluidity and structure by recycling lysophospholipids .
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Exogenous Fatty Acid Uptake: Facilitates the integration of external fatty acids into cellular membranes, enhancing survival under stress .
Recombinant Production
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Expression System: E. coli strains optimized for high-yield protein synthesis .
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Purification: Affinity chromatography (His-tag) followed by lyophilization for long-term storage .
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Quality Control: SDS-PAGE confirms >90% purity, with no contaminating proteins detected .
Experimental Uses
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Enzymatic Assays: Studying lipid acylation kinetics and substrate specificity .
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Membrane Biology Research: Investigating lipid remodeling pathways in E. coli .
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Antimicrobial Target Studies: Exploring vulnerabilities in lipid metabolism for therapeutic development.
Gene Cloning and Overexpression
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Cloning: The aas gene was cloned into plasmid vectors, confirming its role in encoding both acyltransferase and synthetase activities .
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Overexpression: Strains with multiple aas copies showed elevated enzymatic activity and enhanced fatty acid incorporation into membranes .
Enzyme Activity Characterization
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Substrate Specificity: Preferentially uses acyl-ACP as a fatty acid donor for 2-acyl-GPE .
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Kinetic Parameters: Optimal activity under physiological pH and ATP/Mg²⁺ conditions .
References
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your preferred format in order notes for customized preparation.
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The tag type is determined during production. If you require a specific tag, please inform us, and we will prioritize its development.
Target Background
This bifunctional protein plays a crucial role in lysophospholipid acylation. Specifically, it catalyzes the transfer of fatty acids to the 1-position of lysophospholipids via an enzyme-bound acyl-ACP intermediate, requiring ATP and magnesium. Its physiological function is the regeneration of phosphatidylethanolamine from 2-acyl-glycero-3-phosphoethanolamine (2-acyl-GPE), a byproduct of transacylation reactions or phospholipase A1 degradation.
KEGG: ecf:ECH74115_4103
Q&A
What is the Bifunctional protein aas(aas) from E. coli O157:H7?
The Bifunctional protein aas(aas) from E. coli O157:H7 is a 719-amino acid protein (UniProt: B5Z4F4) with two functional domains, primarily involved in phospholipid metabolism . It functions as a 2-acylglycerophosphoethanolamine acyltransferase (EC 2.3.1.40) and interacts with acyl-carrier proteins and phospholipids . The protein has a molecular weight of 67 kDa in native E. coli O157:H7 and 78 kDa when expressed in laboratory E. coli strains, suggesting potential structural or processing differences between native and recombinant forms . It is encoded by the aas gene, with the locus name ECH74115_4103 in the E. coli O157:H7 strain EC4115 genome .
How should recombinant aas protein be stored to maintain activity?
Based on manufacturer recommendations, the following storage conditions are optimal for maintaining recombinant aas protein activity :
| Storage Purpose | Recommended Conditions |
|---|---|
| Long-term storage | -20°C to -80°C in aliquots to avoid freeze-thaw cycles |
| Working solutions | 4°C for up to one week |
| Buffer composition | Tris-based buffer with 50% glycerol, pH 8.0 |
| Reconstitution | Deionized sterile water to 0.1-1.0 mg/mL |
| Shipping form | Lyophilized powder or solution in stabilizing buffer |
Repeated freeze-thaw cycles should be strictly avoided as they significantly reduce protein activity. For applications requiring high enzymatic activity, researchers should validate the activity of each lot using appropriate enzyme assays before proceeding with experiments .
What expression systems are optimal for producing recombinant E. coli O157:H7 aas protein?
The most effective expression system for recombinant aas protein has been E. coli-based systems, with several considerations for optimal yield and activity :
| Expression Component | Recommended Approach |
|---|---|
| Expression host | E. coli BL21(DE3) or similar strains optimized for protein expression |
| Vector type | pET-based vectors with inducible T7 promoter |
| Tags | N-terminal His-tag for simplified purification |
| Induction conditions | IPTG at 0.1-0.5 mM, 16-20°C for 18-24 hours to enhance solubility |
| Growth media | Enriched media (e.g., TB or 2xYT) supplemented with appropriate antibiotics |
| Co-expression | Consider chaperone co-expression to enhance folding |
Researchers should note that membrane-associated proteins like aas often require specialized expression conditions to maintain solubility and proper folding. Lower induction temperatures and longer induction times have been shown to improve the yield of correctly folded protein .
What purification strategy yields the highest purity and activity for recombinant aas protein?
A multi-step purification strategy is recommended to achieve high purity while maintaining enzymatic activity :
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Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged protein
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Intermediate purification: Ion exchange chromatography to remove remaining contaminants
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Polishing step: Size exclusion chromatography to separate aggregates and ensure homogeneity
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Buffer optimization: Final buffer exchange into Tris-based buffer with 50% glycerol at pH 8.0
Key buffer considerations include:
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Addition of reducing agents (1-5 mM DTT or β-mercaptoethanol) to prevent oxidation
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Inclusion of protease inhibitors during initial lysis steps
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Maintaining physiological salt concentrations (150-300 mM NaCl)
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Possible addition of mild detergents (0.01-0.05% non-ionic) if solubility issues arise
The purified protein should achieve >90% purity as determined by SDS-PAGE and maintain enzymatic activity, which should be verified through appropriate activity assays .
What enzymatic assays can be used to characterize the dual functions of the aas protein?
The bifunctional nature of the aas protein requires specific assays for each domain :
For 2-acylglycerophosphoethanolamine acyltransferase activity (EC 2.3.1.40):
| Assay Type | Methodology | Detection Method | Sensitivity |
|---|---|---|---|
| Radiometric | [14C]- or [3H]-labeled acyl donors | Scintillation counting | High (pmol range) |
| Spectrophotometric | Coupled enzyme assay with release of chromogenic/fluorogenic group | Absorbance/fluorescence | Moderate (nmol range) |
| LC-MS/MS | Detection of reaction products | Mass spectrometry | High (pmol range) |
| Colorimetric | Detection of free thiol groups from released CoA | Absorbance | Moderate (nmol range) |
For second domain (acyl-carrier-protein interaction):
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Protein-protein interaction assays (e.g., pull-down assays, surface plasmon resonance)
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Fluorescence-based binding assays using labeled acyl-carrier proteins
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Isothermal titration calorimetry to determine binding kinetics
Researchers should validate these assays with appropriate positive and negative controls, and consider the impact of buffer conditions, pH, and temperature on enzyme activity .
How can site-directed mutagenesis be used to study the catalytic mechanisms of aas protein?
Site-directed mutagenesis provides a powerful approach to understand structure-function relationships in the aas protein :
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Target residue identification: Based on sequence homology with related enzymes, identify potential catalytic residues (serine, histidine, or aspartate in the active site of acyltransferases)
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Mutagenesis strategy: Use overlap extension PCR mutagenesis to create point mutations (e.g., S→A, H→A, D→A) in the predicted catalytic residues
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Mutation verification: Sequence the entire gene to confirm the desired mutation and absence of additional mutations
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Expression and purification: Express and purify the mutant proteins using the same protocols as for wild-type
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Activity comparison: Measure the enzymatic activities of wild-type and mutant proteins under identical conditions
| Predicted Function | Target Residues | Recommended Mutations | Expected Outcome |
|---|---|---|---|
| Catalytic nucleophile | Serine residues | S→A substitution | Loss of acyltransferase activity |
| Active site base | Histidine residues | H→A substitution | Reduced catalytic efficiency |
| Substrate binding | Arginine/lysine residues | R→A or K→A substitution | Altered substrate specificity |
Researchers should complement these functional studies with structural analyses to fully understand the impact of mutations on protein conformation and substrate binding .
How does the aas protein contribute to E. coli O157:H7 pathogenicity and survival in host environments?
Though the direct role of aas in pathogenicity is not fully characterized, several potential contributions can be inferred based on its function and E. coli O157:H7 virulence mechanisms :
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Membrane remodeling for environmental adaptation: The phospholipid-modifying activity of aas likely contributes to membrane fluidity adjustments in response to environmental stresses encountered during infection, including pH changes and antimicrobial compounds .
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Acid resistance: E. coli O157:H7 possesses exceptional acid resistance mechanisms that enable survival in the highly acidic stomach environment. As a membrane-associated protein, aas may contribute to maintaining membrane integrity under acidic conditions, supporting the bacterium's low infectious dose (10-100 CFU) .
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Nutrient acquisition during infection: By recycling and remodeling membrane phospholipids, aas may help conserve fatty acids and adapt to nutrient limitations in the host environment .
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Potential interaction with host factors: Other E. coli O157:H7 membrane proteins have been shown to interact with host components. For example, the Slp (carbon starvation-inducible lipoprotein) interacts with the human polymeric immunoglobulin receptor (pIgR) . Similar interactions involving aas remain to be investigated.
The role of aas should be studied in the context of E. coli O157:H7's comprehensive virulence mechanisms, including Shiga toxin production, type III secretion systems, and other membrane-associated virulence factors .
How has the aas gene evolved in the transition from E. coli O55:H7 to the more virulent O157:H7?
The evolution of E. coli O157:H7 from its O55:H7 ancestor provides context for understanding potential changes in genes like aas :
| Evolutionary Aspect | Findings from Comparative Genomics | Implications for aas |
|---|---|---|
| Mutation rate | 50% more synonymous mutations in O157:H7 vs. O55:H7 | Potentially accelerated evolution of genes including aas |
| Genomic acquisitions | 23 phage genomes/elements in O157:H7 vs. 19 in O55:H7 | Increased horizontal gene transfer potential affecting gene neighborhoods |
| Divergence timeline | Estimated at ~400 years using new molecular clock calculations | Relatively recent adaptations to new ecological niches |
| Selective pressures | More changes observed in O157:H7 lineages | Potential selection for enhanced membrane adaptation functions |
Genomic context analysis of the aas gene might reveal whether it was subject to horizontal gene transfer, selective pressure, or regulatory changes during this evolutionary transition. The significant genomic differences between these closely related strains suggest that many genes, potentially including aas, may have adaptively evolved to enhance virulence and environmental persistence in E. coli O157:H7 .
What CRISPR-Cas9 approaches can be used to study aas function in E. coli O157:H7?
CRISPR-Cas9 technology offers several sophisticated approaches to study aas function in E. coli O157:H7:
Technical considerations for CRISPR applications in E. coli O157:H7 include:
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Selection of appropriate Cas9 variants (SpCas9, SaCas9) based on PAM site availability
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Efficient delivery methods (electroporation of ribonucleoprotein complexes)
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Screening methods (antibiotic selection, colony PCR, sequencing)
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Potential off-target effects and strategies to minimize them
These approaches would provide unprecedented insights into aas function while maintaining the native genomic context .
What structural approaches can reveal the catalytic mechanisms of the bifunctional aas protein?
Understanding the structural basis of aas function requires multiple complementary approaches:
For membrane-associated proteins like aas, structural studies may require:
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Detergent screening to identify optimal solubilization conditions
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Lipid nanodiscs or bicelles to provide a native-like membrane environment
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Truncation constructs focusing on individual domains if the full-length protein proves recalcitrant
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Co-crystallization with substrates, products, or inhibitors to capture functionally relevant states
These structural insights would significantly advance understanding of how aas coordinates its dual enzymatic activities and interacts with membrane components .
How do post-translational modifications affect the structure and function of native versus recombinant aas protein?
The difference in molecular weight between native aas in E. coli O157:H7 (67 kDa) and recombinant aas in laboratory E. coli strains (78 kDa) suggests important post-translational differences :
| Potential Modification | Detection Method | Functional Implication | Recombinant Strategy to Address |
|---|---|---|---|
| Proteolytic processing | N-terminal sequencing, mass spectrometry | May remove regulatory domains or signal sequences | Express with removable tags in positions that don't interfere with processing |
| Lipidation (N-acylation) | Mass spectrometry with lipid-specific detection | Membrane anchoring and localization | Co-expression with native modification enzymes |
| Phosphorylation | Phospho-specific antibodies, MS/MS | Regulation of enzymatic activity | In vitro phosphorylation with kinases, phosphomimetic mutations |
| Conformational differences | Circular dichroism, limited proteolysis | Altered substrate accessibility | Expression in presence of membrane-mimetic environments |
A comprehensive analysis would require:
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Purification of native aas from E. coli O157:H7 under non-denaturing conditions
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Comparative mass spectrometry profiling against recombinant versions
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Activity assays comparing native and recombinant forms
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Structural analysis to detect conformational differences
These studies would help researchers develop expression and purification strategies that yield recombinant aas protein more closely resembling the native form .
How can recombinant aas protein be utilized in developing detection methods for E. coli O157:H7?
The unique properties of the aas protein offer several approaches for developing E. coli O157:H7 detection systems :
| Detection Strategy | Methodology | Sensitivity Range | Application Context |
|---|---|---|---|
| Antibody-based biosensors | Generation of anti-aas antibodies for ELISA or lateral flow assays | 10²-10⁵ CFU/mL | Field-deployable detection kits |
| Bifunctional protein complex biosensors | Engineering aas fusion with reporter proteins (GFP, luciferase) | 10-10³ CFU/mL | Highly sensitive laboratory diagnostics |
| Aptamer-based detection | Selection of DNA/RNA aptamers targeting unique aas epitopes | 10-10⁴ CFU/mL | Rapid, reagent-free detection systems |
| Enzymatic activity assays | Detecting aas-specific enzymatic activity in environmental samples | 10³-10⁶ CFU/mL | Functional detection in complex matrices |
The bifunctional protein complex approach described in search result could be particularly promising, as it notes: "Due to the substantial loading of functional components within the complex, the proposed biosensor demonstrates a simple procedure and high sensitivity."
Development considerations include:
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Cross-reactivity testing against other bacterial species
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Stability testing for field deployment
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Validation using standard detection methods like culture and PCR
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Optimization for various sample types (food, water, clinical samples)
These detection methods could complement existing protocols for E. coli O157:H7 identification, such as the Environmental Protection Agency's "Standard Analytical Protocol for Escherichia coli O157:H7 in Water" .
What role might aas inhibitors play in developing novel antimicrobial strategies against E. coli O157:H7?
Targeting the aas protein could provide a novel approach for antimicrobial development, especially given current challenges in treating E. coli O157:H7 infections :
| Therapeutic Strategy | Mechanism of Action | Potential Advantages | Research Challenges |
|---|---|---|---|
| Competitive substrate analogs | Block active sites of aas enzymatic domains | Potentially specific to bacterial phospholipid metabolism | Designing compounds with suitable membrane permeability |
| Allosteric inhibitors | Disrupt conformational changes required for activity | May offer higher specificity than active site inhibitors | Identifying suitable allosteric binding sites |
| Domain interface disruptors | Prevent coordination between the two functional domains | Novel mechanism distinct from traditional antibiotics | Complex design requirements for targeting protein-protein interfaces |
| Anti-virulence approach | Disrupt membrane homeostasis without direct bacterial killing | Potentially lower selection pressure for resistance | Demonstrating efficacy in infection models |
Development considerations include:
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Screening assay development using purified recombinant aas
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Structure-based drug design informed by crystallographic or modeling data
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Counter-screening against human homologs to minimize toxicity
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Evaluation in animal models of E. coli O157:H7 infection
The appeal of this approach is reinforced by search result , which notes: "Treating E. coli O157:H7 infection with antimicrobial agents is associated with an increased risk of severe sequelae such as HUS. The difficulty in treating this bacterium using conventional modalities of antimicrobial agent administration has sparked an interest in investigating new therapeutic approaches to this bacterium."
