Recombinant Escherichia coli UPF0721 transmembrane protein yfcA (yfcA)
Description
Introduction to UPF0721 Transmembrane Protein yfcA
The UPF0721 transmembrane protein yfcA is classified as an integral membrane protein that spans the entire cell membrane of Escherichia coli. The designation "UPF" (Uncharacterized Protein Family) indicates that while the protein has been identified and sequenced, its precise biological function remains incompletely characterized. This protein belongs to the broader category of transmembrane proteins, which function as critical gateways that permit the transport of specific substances across cellular membranes . Like other transmembrane proteins, yfcA likely undergoes significant conformational changes to facilitate the movement of substances through the membrane, a characteristic feature of transport proteins .
The yfcA protein is encoded by the yfcA gene found in various E. coli strains, including pathogenic variants such as E. coli O157:H7. In commercial contexts, this protein is available as a recombinant product expressed in E. coli expression systems, typically with affinity tags to facilitate purification and downstream applications . The full-length protein consists of 269 amino acids and has been assigned UniProt identification numbers P0AD30 and P0AD32, depending on the specific E. coli strain .
Recombinant Protein Products
Recombinant UPF0721 transmembrane protein yfcA is commercially available from several biotechnology suppliers, provided as purified protein preparations for research applications. The table below summarizes the key specifications of commercially available recombinant yfcA protein products:
Predicted Function as a Membrane Transporter
While the exact function of UPF0721 transmembrane protein yfcA has not been fully characterized, its classification and structural features provide important clues about its potential biological role. The protein is annotated as a "Probable membrane transporter protein YfcA" in protein databases, suggesting its involvement in the movement of specific molecules across the bacterial cell membrane .
As a transmembrane protein, yfcA likely functions as a gateway that permits the transport of specific substances across the membrane . Like other transport proteins, it may undergo significant conformational changes to facilitate the movement of its substrates through the membrane barrier. The specific substrates transported by yfcA have not been definitively identified in the available research literature, representing an area for future investigation.
Role in Bacterial Physiology
The presence of yfcA across various E. coli strains, including pathogenic variants like E. coli O157:H7, suggests potential importance in bacterial physiology and possibly in pathogenesis. Transmembrane transporters play crucial roles in nutrient acquisition, waste elimination, cell signaling, and maintaining cellular homeostasis. The conservation of this protein could indicate an essential function in bacterial survival or adaptation to specific environmental conditions.
While not directly related to yfcA, research on E. coli has identified operons containing multiple genes required for functions such as capsule formation, which involves transmembrane transport systems . Such studies highlight the importance of membrane proteins in bacterial cell envelope biogenesis and potentially in virulence mechanisms of pathogenic strains.
Immunological Applications
Purified recombinant yfcA protein can be used for the development of antibodies specific to this transmembrane protein. Such antibodies could serve as valuable tools for localization studies, expression analysis, and detection of yfcA in various bacterial samples. Furthermore, the protein can be employed in enzyme-linked immunosorbent assays (ELISA) as evidenced by available ELISA recombinant yfcA products .
Potential Therapeutic and Biotechnological Applications
As a bacterial membrane protein, yfcA presents a potential target for antimicrobial development, particularly if future research establishes its essential role in bacterial physiology or pathogenesis. Membrane proteins are increasingly recognized as valuable targets for antibiotic development due to their accessibility and critical functions.
From a biotechnological perspective, understanding the structure and function of membrane transporters like yfcA could inform the development of engineered transport systems for applications in bioremediation, biosensing, or bioproduction processes. Bacterial transmembrane proteins have found applications in synthetic biology approaches aiming to create cells with novel transport capabilities or sensing functions.
Product Specs
Note: We will prioritize shipping the format we have in stock. However, if you have a specific format requirement, please indicate it when placing your order, and we will fulfill your request.
Note: All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance, as additional fees will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Target Background
KEGG: ecj:JW2324
STRING: 316385.ECDH10B_2489
Q&A
What expression systems are recommended for recombinant yfcA production?
E. coli remains the preferred expression system for recombinant yfcA production due to its rapid growth, high protein yields, and relatively low cost . For optimal expression, consider the following methodological approaches:
| Expression System Component | Recommended Options | Considerations |
|---|---|---|
| E. coli Strain | BL21(DE3), C41(DE3), C43(DE3) | C41 and C43 are specifically engineered for membrane protein expression |
| Expression Vector | pET with N-terminal His-tag | Facilitates purification while minimizing interference with transmembrane domains |
| Induction | IPTG at 0.1-0.5 mM | Lower concentrations and lower temperatures (16-25°C) often yield more soluble protein |
| Growth Media | Terrific Broth or 2xYT | Rich media support higher cell densities and protein yields |
When expressing membrane proteins like yfcA, slower induction rates and lower growth temperatures often yield better results by allowing proper membrane integration rather than inclusion body formation .
How should researchers troubleshoot inclusion body formation when expressing yfcA?
Inclusion body formation is a common challenge when expressing membrane proteins like yfcA in E. coli. To overcome this issue, implement a systematic troubleshooting approach:
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Reduce expression rate by lowering the incubation temperature to 16-25°C after induction
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Decrease inducer concentration (0.1-0.2 mM IPTG rather than 1 mM)
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Use specialty E. coli strains (C41, C43) engineered for membrane protein expression
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Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ)
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Add mild detergents (0.1-0.5% Triton X-100) to culture media to aid solubilization
For analytical assessment, always run parallel small-scale expression tests with varying conditions before scaling up to production levels .
What purification strategies are effective for recombinant yfcA protein?
Purification of membrane proteins like yfcA requires specialized techniques:
| Purification Step | Methodology | Critical Parameters |
|---|---|---|
| Cell Lysis | French press or sonication | Gentle disruption to preserve membrane structures |
| Membrane Isolation | Ultracentrifugation | 100,000×g, 1 hour to isolate membrane fractions |
| Solubilization | Detergent extraction | n-Dodecyl β-D-maltoside (DDM) or similar at 1-2% |
| IMAC Purification | Ni-NTA chromatography | Use detergent-containing buffers (0.1-0.2% DDM) |
| Buffer Exchange | Size exclusion chromatography | Remove excess detergent and imidazole |
Following purification, protein purity should be confirmed via SDS-PAGE, with expected purity greater than 90% . For functional studies, verify proper folding using circular dichroism spectroscopy.
What reconstitution methods are recommended for lyophilized yfcA protein?
When working with lyophilized yfcA preparations, proper reconstitution is critical for maintaining protein structure and function:
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Centrifuge the vial briefly to collect all material at 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% to prevent freeze-thaw damage
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For membrane protein functionality, consider reconstitution into liposomes or nanodiscs
Avoid repeated freeze-thaw cycles, which can significantly decrease protein activity. For long-term storage, prepare small aliquots with 50% glycerol and store at -20°C/-80°C .
How can systems biology approaches enhance understanding of yfcA function?
Systems biology offers powerful tools for investigating membrane transporters like yfcA:
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Transcriptomics: Analyze gene expression profiles under different conditions to identify co-regulated genes that might function in the same pathway as yfcA
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Proteomics: Use pull-down assays with tagged yfcA to identify interaction partners
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Metabolomics: Compare metabolite profiles in wild-type versus yfcA knockout strains to identify potential transported substrates
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Flux balance analysis: Develop computational models to predict the impact of yfcA activity on cellular metabolism
These approaches should be complemented with targeted biochemical assays to validate predictions . When reporting results, include comprehensive metadata about experimental conditions to ensure reproducibility.
What bioinformatic approaches can predict substrates and mechanism of the yfcA transporter?
Predicting the substrates and transport mechanism of yfcA requires integrated bioinformatic analysis:
| Analytical Approach | Tools/Methods | Expected Insights |
|---|---|---|
| Homology Modeling | AlphaFold2, SWISS-MODEL | 3D structural predictions |
| Molecular Dynamics | GROMACS, NAMD | Conformational changes during transport |
| Evolutionary Analysis | ConSurf, Clustal Omega | Conserved functional residues |
| Substrate Docking | AutoDock Vina, HADDOCK | Potential binding sites and substrates |
| Genomic Context | STRING, EcoCyc | Functional associations with metabolic pathways |
Begin with sequence-based comparisons to characterized transporters, then progress to more sophisticated structural analyses. Integration of these approaches with experimental validation (site-directed mutagenesis, transport assays) provides the most robust functional predictions .
What experimental approaches can determine the topology and membrane integration of yfcA?
Determining the precise membrane topology of yfcA is essential for understanding its function:
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Cysteine scanning mutagenesis: Replace endogenous cysteines and introduce single cysteines at specific positions, then test their accessibility to membrane-impermeable thiol-reactive reagents
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Fusion reporter systems: Create fusion constructs with reporters like GFP, PhoA, or LacZ at various truncation points to determine cytoplasmic vs. periplasmic orientation
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Protease protection assays: Expose membrane preparations to proteases with/without detergent solubilization to identify protected domains
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Cryo-electron microscopy: For high-resolution structural determination in native-like environments
Data interpretation should integrate computational predictions with experimental results to develop a comprehensive topological model.
How can researchers optimize conditions to maintain yfcA stability during functional studies?
Maintaining stability of membrane proteins like yfcA during functional characterization requires careful optimization:
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Detergent screening: Systematically test multiple detergent types (maltosides, glucosides, neopentyl glycols) at various concentrations
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Lipid supplementation: Add specific phospholipids (PE, PG, cardiolipin) found in E. coli membranes to mimic native environment
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Buffer optimization: Test various pH conditions (6.5-8.0), salt concentrations (100-500 mM NaCl), and stabilizing additives (glycerol, trehalose)
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Thermal stability assays: Employ differential scanning fluorimetry to quantitatively assess protein stability under different conditions
Researchers should report comprehensive stability data alongside functional measurements to ensure reproducibility of results across laboratories .
What strategies can improve soluble expression yields of yfcA for structural studies?
For structural biology applications requiring high yields of properly folded yfcA:
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Fusion partners: Incorporate solubility-enhancing tags such as MBP, SUMO, or Fh8
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Codon optimization: Adjust codon usage to match E. coli preferences, particularly for rare codons
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Periplasmic targeting: Direct expression to the periplasm using appropriate signal sequences
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Cell-free expression: Consider E. coli extract-based cell-free systems with added lipids or detergents
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Host strain engineering: Use strains with enhanced membrane protein expression capacity or modified stress responses
Monitor expression using fluorescent fusion proteins to rapidly identify optimal conditions before scaling up production . Systematic exploration of these parameters using a Design of Experiments (DoE) approach can efficiently identify optimal expression conditions.
What spectroscopic methods are suitable for studying yfcA conformational changes?
Several spectroscopic techniques can provide insights into yfcA conformational dynamics:
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Circular Dichroism (CD): Assess secondary structure content and changes upon substrate binding
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Fluorescence Spectroscopy: Utilize intrinsic tryptophan fluorescence or introduced fluorescent labels to monitor conformational changes
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Fourier Transform Infrared Spectroscopy (FTIR): Analyze hydrogen-deuterium exchange to identify accessibility changes during transport cycles
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Nuclear Magnetic Resonance (NMR): For residue-specific structural analysis of isotope-labeled protein
Researchers should begin with CD to confirm proper folding before proceeding to more sophisticated analyses. Changes in spectroscopic signatures upon addition of potential substrates can provide initial evidence of binding interactions.
How can genetic approaches be used to elucidate yfcA function in vivo?
Genetic manipulation provides powerful tools to investigate yfcA function:
| Genetic Approach | Methodology | Expected Outcome |
|---|---|---|
| Gene Knockout | CRISPR-Cas9 or λ-Red Recombination | Phenotypic consequences of yfcA absence |
| Complementation | Plasmid-based expression in knockout strain | Confirmation of phenotype specificity |
| Site-directed Mutagenesis | Alanine scanning of conserved residues | Identification of functional residues |
| Suppressor Screens | Selection for mutations restoring function | Identification of interacting pathways |
| Conditional Expression | Titratable promoters (tetR system) | Dose-response relationship between yfcA levels and function |
Analysis should include growth rate measurements, metabolic profiling, and stress response assessment under various environmental conditions to identify the physiological role of yfcA .
What transport assay methods can determine yfcA substrate specificity?
To identify substrates transported by yfcA:
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Whole-cell uptake assays: Measure accumulation of radiolabeled or fluorescent potential substrates in cells overexpressing yfcA compared to control cells
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Liposome reconstitution: Purify yfcA and reconstitute into liposomes loaded with potential substrates to measure transport rates
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Electrophysiological methods: Use patch-clamp techniques with yfcA reconstituted into giant liposomes or planar lipid bilayers to measure transport-associated currents
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Counterflow assays: Pre-load liposomes with unlabeled substrate and measure uptake of labeled substrate in exchange
When designing transport assays, account for the possibility of symport, antiport, or uniport mechanisms, and test various counter-ions and membrane potential conditions.
