Recombinant Escherichia coli O45:K1 Bifunctional protein aas (aas)
Product Specs
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Target Background
This protein plays a crucial role in lysophospholipid acylation. 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 involves the regeneration of phosphatidylethanolamine from 2-acyl-glycero-3-phosphoethanolamine (2-acyl-GPE), a byproduct of transacylation reactions or phospholipase A1 degradation.
KEGG: ecz:ECS88_3131
Q&A
What is the Bifunctional protein aas(aas) and what is its significance in research?
The Bifunctional protein aas(aas) from Escherichia coli O45:K1 (UniProt ID: B7MLI2) is a full-length 719 amino acid protein involved in bacterial metabolism. The protein contains multiple functional domains that participate in fatty acid metabolism pathways, making it valuable for studies of bacterial physiology and potential antimicrobial targeting. The recombinant form, typically expressed with an N-terminal His tag, provides researchers with a purified version for in vitro studies of enzyme kinetics, structure-function relationships, and pathway analysis .
What expression systems are suitable for producing recombinant Bifunctional protein aas(aas)?
E. coli expression systems are primarily used for producing recombinant Bifunctional protein aas(aas), as demonstrated in the commercial preparation where the protein is expressed in E. coli . For optimal expression, researchers should consider:
| Expression System | Advantages | Considerations |
|---|---|---|
| E. coli BL21(DE3) | High yield, cost-effective, rapid growth | Potential inclusion body formation |
| E. coli Rosetta™ | Enhanced expression of proteins with rare codons | Higher cost than standard strains |
| E. coli Origami™ | Promotes disulfide bond formation | Slower growth rate |
| Cell-free systems | Avoids toxicity issues, rapid production | Lower yield, higher cost |
For most applications, standard E. coli expression using the pET vector system with IPTG induction provides sufficient yields of active protein, especially when including optimization steps for temperature, induction time, and media composition.
What are the optimal conditions for expression of recombinant Bifunctional protein aas(aas) in E. coli?
Optimizing expression conditions is critical for obtaining high yields of functional protein. For Bifunctional protein aas(aas), consider the following parameters:
| Parameter | Recommended Conditions | Rationale |
|---|---|---|
| Temperature | 16-25°C post-induction | Reduces inclusion body formation |
| Induction OD₆₀₀ | 0.6-0.8 | Balances cell density and protein synthesis capacity |
| IPTG concentration | 0.1-0.5 mM | Lower concentrations reduce toxicity while maintaining expression |
| Expression time | 16-20 hours | Extended time allows protein accumulation at lower temperatures |
| Media | TB or 2×YT with supplemental glucose | Rich media supports higher cell density and protein yield |
For proteins prone to aggregation, like many bacterial enzymes, lower induction temperatures significantly improve the proportion of soluble protein. Post-induction optimization experiments should be conducted to determine the specific conditions that maximize yield of active protein rather than focusing solely on total expression levels .
How can codon optimization improve the expression of Bifunctional protein aas(aas)?
Codon optimization can significantly enhance expression levels, particularly for bacterial proteins expressed in heterologous systems. For Bifunctional protein aas(aas):
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Analyze the coding sequence for rare codons using tools like the Codon Usage Database or OPTIMIZER
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Replace rare codons with synonymous codons common in the expression host
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Adjust the GC content to match the expression host's preference
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Eliminate potential mRNA secondary structures, particularly in the 5' region
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Consider using specialized strains like Rosetta that supply additional tRNAs for rare codons
What strategies can prevent protein aggregation during expression of Bifunctional protein aas(aas)?
Preventing protein aggregation is essential for obtaining functional Bifunctional protein aas(aas). Research has shown that E. coli cells naturally segregate protein aggregates asymmetrically to older poles during cell division . To leverage cellular mechanisms and minimize aggregation:
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Reduce expression rate by lowering temperature (16-18°C) and inducer concentration
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Co-express molecular chaperones (GroEL/ES, DnaK/J) to assist proper folding
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Include solubility-enhancing fusion partners (MBP, SUMO, Trx) in the expression construct
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Add low concentrations (1-5%) of solubilizing agents like glycerol or sorbitol to the culture medium
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Use E. coli strains engineered for improved protein folding (e.g., SHuffle, Origami)
For Bifunctional protein aas(aas), maintaining the native protein structure is critical for preserving enzymatic activity. Microscopic analysis of protein distribution during expression can help identify optimal conditions that minimize aggregation while maximizing functional protein yield.
What purification strategies optimize yield and maintain activity of Bifunctional protein aas(aas)?
The purification strategy should be designed to maximize both yield and activity of the recombinant protein:
For His-tagged Bifunctional protein aas(aas), immobilized metal affinity chromatography (IMAC) using Ni-NTA resin provides high selectivity as the initial capture step. For applications requiring higher purity, additional chromatographic steps should be incorporated. Throughout purification, monitor enzymatic activity to ensure the purification process preserves the functional integrity of the protein.
What protein assays are most suitable for quantifying recombinant Bifunctional protein aas(aas)?
Selecting appropriate quantification methods ensures accurate determination of protein concentration:
| Assay Method | Advantages | Limitations |
|---|---|---|
| Bradford Assay | Rapid, minimal interference from nucleic acids | Variable response to different proteins |
| BCA Assay | Compatible with many detergents, high sensitivity | Interference from reducing agents |
| UV Absorbance (A₂₈₀) | Non-destructive, rapid | Interference from nucleic acids |
| SDS-PAGE with densitometry | Visual confirmation of purity | Semi-quantitative |
For routine quantification of purified Bifunctional protein aas(aas), A₂₈₀ measurement provides a convenient approach, especially when the extinction coefficient is known. For more complex samples or when higher accuracy is required, colorimetric assays like Bradford or BCA are recommended, with BSA as the standard protein .
What are the recommended reconstitution procedures for lyophilized Bifunctional protein aas(aas)?
Proper reconstitution is essential for maintaining the functional integrity of lyophilized proteins:
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Centrifuge the vial briefly before opening to bring contents to the bottom
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Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL
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Add glycerol to a final concentration of 5-50% (recommended 50%)
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Aliquot for long-term storage at -20°C/-80°C to avoid repeated freeze-thaw cycles
When reconstituting Bifunctional protein aas(aas), gentle mixing rather than vigorous vortexing helps preserve protein structure and activity. The inclusion of glycerol serves as a cryoprotectant that prevents ice crystal formation and protein denaturation during freezing cycles.
How can I validate the functional activity of recombinant Bifunctional protein aas(aas)?
Validating functional activity ensures that the recombinant protein retains its native enzymatic capabilities:
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Substrate conversion assay: Monitor the conversion of specific substrates using HPLC or coupled enzyme assays
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Binding assays: Assess interaction with natural substrates using isothermal titration calorimetry (ITC)
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Thermal shift assay: Evaluate protein stability in the presence and absence of substrates
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Circular dichroism: Confirm proper secondary structure folding
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Enzymatic activity comparison with native protein (if available)
For Bifunctional protein aas(aas), which is involved in fatty acid metabolism, functional validation might include assessing both its acyl-acyl carrier protein synthetase and 2-acylglycerophosphoethanolamine acyltransferase activities through specific substrate conversion assays.
What experimental approaches can be used to study protein-protein interactions involving Bifunctional protein aas(aas)?
Understanding protein-protein interactions provides insights into biological function and regulatory mechanisms:
| Method | Application | Advantages |
|---|---|---|
| Bacterial two-hybrid | In vivo screening | Identifies novel interaction partners |
| Pull-down assays | In vitro confirmation | Directly demonstrates physical interaction |
| Surface plasmon resonance | Kinetic analysis | Provides binding constants and kinetics |
| Crosslinking coupled with MS | Complex identification | Captures transient interactions |
| FRET/BRET | Real-time monitoring | Allows visualization in living cells |
For E. coli proteins, in vivo approaches like bacterial two-hybrid systems are particularly valuable for identifying physiologically relevant interactions. Techniques like that described in search result for following protein complexes in living cells using fluorescent markers can provide insights into the spatial and temporal dynamics of interactions .
How does post-translational modification affect the activity of recombinant Bifunctional protein aas(aas)?
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N-terminal methionine processing
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Disulfide bond formation
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Proteolytic processing
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Limited phosphorylation and acetylation
For Bifunctional protein aas(aas) expressed in E. coli, the most relevant consideration is proper disulfide bond formation if present in the native structure. Expressing the protein in specialized strains with oxidizing cytoplasmic environments (like Origami) can facilitate correct disulfide formation. Analyzing the recombinant protein by mass spectrometry can identify any unexpected modifications that might impact activity.
What are the best methods for long-term storage of recombinant Bifunctional protein aas(aas)?
Proper storage is critical for maintaining protein stability and activity over time:
| Storage Condition | Recommendation | Duration |
|---|---|---|
| Lyophilized state | -20°C/-80°C | Long-term (years) |
| Solution with 50% glycerol | -20°C/-80°C | Long-term (months to years) |
| Working solution | 4°C | Short-term (up to one week) |
For Bifunctional protein aas(aas), the recommended storage buffer is Tris/PBS-based with 6% trehalose at pH 8.0 . Trehalose serves as a stabilizing agent that preserves protein structure during freeze-thaw cycles. Aliquoting the protein solution prevents repeated freeze-thaw cycles, which can lead to progressive denaturation and loss of activity.
How can I troubleshoot low yield or activity of recombinant Bifunctional protein aas(aas)?
When experiencing issues with protein yield or activity, systematic troubleshooting can identify and resolve the underlying causes:
| Issue | Potential Causes | Troubleshooting Approaches |
|---|---|---|
| Low expression | Poor codon optimization, toxic effects | Try different expression strains, reduce induction temperature |
| Insoluble protein | Improper folding, aggregation | Co-express chaperones, use solubility tags, optimize buffer conditions |
| Low activity | Improper folding, inhibitory compounds | Optimize purification protocol, include stabilizing additives |
| Protein degradation | Proteolytic activity | Include protease inhibitors, use protease-deficient strains |
For Bifunctional protein aas(aas), comparing SDS-PAGE analysis before and after purification can help identify at which stage losses occur. Activity assays using positive controls can distinguish between yield issues and activity issues. Microscopic analysis might reveal asymmetric segregation of protein aggregates in E. coli cells, suggesting optimization strategies to enhance soluble expression .
What strategies can improve the solubility of recombinant Bifunctional protein aas(aas)?
Enhancing protein solubility is often key to obtaining functional recombinant proteins:
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Optimize expression conditions (temperature, induction time, media composition)
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Use solubility-enhancing fusion partners (MBP, SUMO, TRX)
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Screen various buffer conditions during purification and storage
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Include stabilizing additives like trehalose (as used in the commercial preparation )
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Consider protein engineering to remove hydrophobic patches or introduce solubilizing mutations
Recent research on protein aggregation in E. coli has shown that cells naturally employ asymmetric strategies to segregate protein aggregates to older cell poles during division . This understanding can guide experimental design, particularly when monitoring expression using fluorescent protein fusions to detect aggregation patterns early in the optimization process.
