Recombinant Salmonella dublin UPF0756 membrane protein YeaL (yeaL)
Description
Introduction to Salmonella dublin and the YeaL Protein
Salmonella dublin is a host-adapted serovar of Salmonella enterica that primarily affects cattle, where it can cause severe disease and significantly impact animal welfare and agricultural economics. This pathogen can rapidly disseminate throughout the host organism after initial intestinal colonization, producing systemic disease and establishing persistent infections in various organs including the spleen, liver, and lungs . The bacterium's ability to survive as a facultative intracellular pathogen enables it to evade host immune responses, contributing to its pathogenicity.
The UPF0756 membrane protein YeaL (yeaL) is one of numerous proteins expressed by Salmonella dublin. The "UPF" designation (Uncharacterized Protein Family) indicates that the precise biological function of this protein family remains to be fully elucidated. As a membrane protein, YeaL is integrated into the bacterial cell membrane, suggesting potential roles in membrane integrity, transport processes, or host-pathogen interactions .
Molecular Classification and Context
YeaL belongs to a family of membrane proteins found across various bacterial species including Salmonella and Escherichia coli. The protein is encoded by the yeaL gene, which in Salmonella dublin (strain CT_02021853) is identified by the ordered locus name SeD_A2077 . The conservation of this protein across different bacterial species suggests it may serve an important functional role, though specific studies detailing its precise function are currently limited in the scientific literature.
Expression Systems and Production Methods
The recombinant form of Salmonella dublin UPF0756 membrane protein YeaL is typically produced using E. coli expression systems. This approach allows for efficient production of the protein with appropriate post-translational modifications. The full-length protein (amino acids 1-148) is commonly expressed with an N-terminal histidine tag to facilitate purification through affinity chromatography .
The recombinant production process typically involves:
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Cloning the yeaL gene into an appropriate expression vector
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Transformation of the construct into E. coli host cells
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Induction of protein expression
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Cell lysis and protein extraction
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Purification through affinity chromatography using the His-tag
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Quality control analysis, typically through SDS-PAGE
The resulting purified protein generally achieves greater than 90% purity as determined by SDS-PAGE analysis .
Reconstitution Protocols
The lyophilized protein should be reconstituted following specific guidelines:
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The vial should be briefly centrifuged prior to opening to ensure all material is at the bottom of the container.
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Reconstitution should be performed using deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL.
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For long-term storage after reconstitution, the addition of glycerol to a final concentration of 5-50% is recommended, with 50% being the standard concentration used by many manufacturers.
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The reconstituted protein should be aliquoted and stored at -20°C or -80°C for long-term storage .
For working solutions, reconstituted protein can be stored at 4°C for up to one week, though this should be minimized to preserve protein integrity .
Putative Functions Based on Protein Family
As a membrane protein belonging to the UPF0756 family, YeaL likely plays a role in membrane-associated processes, though the specific function remains to be fully characterized. Based on its membrane localization and structural features, potential functions may include:
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Membrane integrity and stability
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Small molecule transport
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Signal transduction
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Protein-protein interactions at the membrane interface
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Potential involvement in bacterial virulence or stress responses
Context Within Salmonella Pathogenesis
Salmonella dublin employs various virulence factors to establish infection, including Salmonella Pathogenicity Islands (SPIs), toxins, flagella, fimbriae, and virulence plasmids . While the specific role of YeaL in pathogenesis has not been definitively established in the available literature, membrane proteins often contribute to bacterial adaptation to host environments.
Current Research Applications
The recombinant Salmonella dublin UPF0756 membrane protein YeaL has several potential applications in research settings:
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Immunological Studies: As a bacterial protein, recombinant YeaL can be used to study host immune responses to Salmonella dublin.
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Antibody Production: The purified protein can serve as an antigen for generating specific antibodies that may be useful in diagnostic or research applications.
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Structural Studies: The availability of purified protein enables structural analyses through techniques such as X-ray crystallography or nuclear magnetic resonance spectroscopy.
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Protein-Protein Interaction Studies: Recombinant YeaL can be used in assays designed to identify interaction partners, potentially revealing functional roles.
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ELISA Development: The protein is available in ELISA-compatible formats, suggesting applications in diagnostic test development .
Future Research Directions
Several promising avenues for future research on the Salmonella dublin UPF0756 membrane protein YeaL include:
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Functional Characterization: Determining the specific biological function of YeaL through gene knockout studies, complementation experiments, and phenotypic analyses.
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Host-Pathogen Interaction Studies: Investigating whether YeaL plays a role in Salmonella dublin interactions with host cells or immune components.
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Structural Determination: Resolving the three-dimensional structure of YeaL to gain insights into its function and potential as a therapeutic target.
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Comparative Studies: Examining functional differences between YeaL from Salmonella dublin and homologous proteins from other bacterial species.
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Vaccine Development Potential: Assessing whether YeaL could serve as a component of subunit vaccines against Salmonella dublin infections in cattle .
Product Specs
Note: We prioritize shipping the format currently in stock. However, if you have a specific format preference, please indicate it in your order notes, and we will accommodate 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. Lyophilized form has a shelf life of 12 months at -20°C/-80°C.
Target Background
KEGG: sed:SeD_A2077
Q&A
What is the structural organization of the Salmonella Dublin UPF0756 membrane protein YeaL?
The YeaL protein belongs to the UPF0756 family of integral membrane proteins. While specific structural data for Salmonella Dublin YeaL is limited, membrane proteins generally require specialized techniques for structural elucidation. The protein likely contains multiple transmembrane domains that anchor it within the bacterial cell membrane. According to membrane protein database analyses, proteins of this family typically require detergent solubilization for isolation and study . Structural determination would typically employ methods such as X-ray crystallography, cryo-electron microscopy, or nuclear magnetic resonance spectroscopy, with resolution limits typically set at 10 Å for inclusion in structural databases .
How does YeaL expression vary during different stages of Salmonella Dublin infection?
Expression patterns of YeaL during infection remain to be fully characterized. Salmonella Dublin is known to establish both acute and chronic infections, with the ability to persist as a latent carrier state in cattle . Gene expression studies would need to examine YeaL levels during:
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Initial intestinal colonization
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Systemic dissemination
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Persistent carrier state
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Bacterial shedding phases
The bacterium's ability to transition between these states likely involves differential expression of multiple genes, potentially including membrane proteins like YeaL that may sense environmental changes or contribute to stress responses .
How does the membrane environment affect YeaL protein function and stability?
The membrane environment significantly influences membrane protein function and stability. Recent research in membrane protein biology indicates that the lipid composition surrounding membrane proteins is critical for their proper folding and activity . For YeaL specifically, the following environmental factors would be relevant:
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Lipid composition of the bacterial membrane
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Protein-lipid interactions that may stabilize the protein structure
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Influence of pH and temperature on protein conformation
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Interactions with other membrane components
Yale scientists recently received $10.5 million to explore how the cell membrane environment affects membrane proteins, highlighting the importance of studying proteins in their native lipid environment rather than in isolation . This research direction could inform approaches to studying YeaL function in its natural context.
What is the relationship between YeaL and antimicrobial resistance mechanisms in Salmonella Dublin?
Salmonella Dublin has become one of the most multidrug-resistant (MDR) serotypes, with 98.5% of studied isolates resistant to more than four antimicrobials in recent studies . The most common resistance profile includes resistance to:
| Antimicrobial Class | Resistance Frequency | Example Drugs |
|---|---|---|
| Tetracyclines | 97% | Tetracycline |
| Aminoglycosides | 95% | Streptomycin |
| Folate pathway inhibitors | 96% | Sulfonamides |
| Beta-lactams | 85% | Ampicillin, Cefoxitin |
While the specific contribution of YeaL to this resistance profile is unknown, membrane proteins can function as efflux pumps or modify membrane permeability to antibiotics. Research investigating YeaL's potential role in antimicrobial resistance would need to examine changes in resistance profiles when yeaL expression is altered, and potential interactions with known resistance mechanisms .
How might YeaL contribute to Salmonella Dublin's evasion of host immune responses?
Salmonella Dublin's ability to establish persistent infections and the carrier state suggests sophisticated immune evasion mechanisms. Membrane proteins can play critical roles in this process through:
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Alteration of surface antigens recognized by host antibodies
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Interference with complement deposition
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Modification of bacterial surface structures detected by pattern recognition receptors
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Contribution to intracellular survival pathways
YeaL, as a membrane protein, could potentially participate in one or more of these processes. Salmonella Dublin's pathogenicity involves enhanced intracellular proliferation in both intestinal and extraintestinal tissues , and membrane proteins often contribute to these adaptations through sensing host environments or participating in virulence factor regulation.
What expression systems are most effective for producing recombinant Salmonella Dublin YeaL?
For optimal recombinant expression of membrane proteins like YeaL, several systems should be considered:
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E. coli-based systems:
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BL21(DE3) strain with T7 expression systems
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C41(DE3) and C43(DE3) strains engineered specifically for membrane protein expression
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Use of fusion tags (His, MBP, SUMO) to aid solubility and purification
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Yeast expression systems:
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Pichia pastoris for higher eukaryotic-like membrane environment
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Saccharomyces cerevisiae for ease of genetic manipulation
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Cell-free expression systems:
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Wheat germ extract
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E. coli extract supplemented with lipids or detergents
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Each system requires optimization of induction conditions, temperature, and media composition. For membrane proteins, reduced expression temperatures (16-25°C) often improve proper folding and reduce inclusion body formation .
What purification and solubilization strategies yield functional YeaL protein?
Purification of membrane proteins requires specialized approaches:
| Step | Recommended Approach | Considerations |
|---|---|---|
| Membrane extraction | Mechanical disruption with French press or sonication | Gentle methods preserve protein structure |
| Solubilization | Use of mild detergents (DDM, LMNG, CHAPS) | Detergent screening critical for stability |
| Purification | IMAC for His-tagged constructs | Maintain detergent above CMC |
| Size exclusion | Final polishing and detergent exchange | Assess protein oligomerization state |
The Membrane Protein Data Bank (MPDB) contains statistical information on successful detergents and crystallization conditions that can guide optimization . For functional studies, reconstitution into liposomes or nanodiscs may be necessary to maintain the protein in a native-like lipid environment.
What analytical techniques best characterize YeaL's interaction with other membrane components?
Modern analytical approaches for studying membrane protein interactions include:
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Crosslinking mass spectrometry (XL-MS):
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Identifies proximity relationships between proteins
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Can capture transient interactions
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Förster resonance energy transfer (FRET):
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Measures distances between fluorescently labeled proteins
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Can be performed in living cells
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Cryo-electron microscopy:
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Visualizes protein complexes in near-native states
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Recent advances allow near-atomic resolution
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Native mass spectrometry:
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Preserves non-covalent interactions during analysis
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Determines stoichiometry of complexes
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These techniques align with recent interdisciplinary approaches to membrane protein research, such as those being developed at Yale's Cancer Biology and Nanobiology Institutes, which focus on studying membrane proteins in their natural environments rather than in isolation .
Could YeaL serve as a target for vaccine development against Salmonella Dublin?
YeaL's potential as a vaccine target would depend on several factors:
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Conservation across Salmonella Dublin strains
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Surface exposure and accessibility to antibodies
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Role in virulence or survival
Current vaccination approaches for Salmonella Dublin include commercial live culture vaccines administered to carrier cows. One study demonstrated that vaccinating S. Dublin latent carrier cows at dry-off reduced intrauterine transmission to calves . The efficacy of such approaches indicates that targeting virulence or survival factors can be productive vaccine strategies.
A protein-based vaccine approach would require:
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Identification of immunogenic epitopes
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Demonstration of protective immunity in animal models
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Comparison with existing vaccine approaches
Research would need to establish whether antibodies against YeaL could neutralize bacterial function or enhance clearance by the immune system.
How might genomic variation in yeaL correlate with Salmonella Dublin virulence across different isolates?
Genomic analysis across Salmonella Dublin isolates could reveal:
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Sequence variation in the yeaL gene
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Correlation between specific yeaL variants and virulence phenotypes
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Evidence of selective pressure on the gene during host adaptation
Salmonella Dublin has demonstrated increasing prevalence and antimicrobial resistance in dairy facilities . Genomic characterization studies have identified virulence factors including Salmonella Pathogenicity Islands (SPI-1, SPI-2, SPI-6, and SPI-19) and virulence plasmids like pSDV containing the spv operon .
Comparative genomic analysis would need to examine whether yeaL sequence variations correlate with:
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Host adaptation capabilities
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Antimicrobial resistance profiles
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Persistence in carrier animals
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Transmission efficiency
Such analysis could help determine if YeaL contributes to the reported increase in prevalence of Salmonella Dublin in the U.S. dairy industry .
