Recombinant Erwinia tasmaniensis UPF0756 membrane protein ETA_17460 (ETA_17460)
Description
Protein Identity and Classification
The UPF0756 membrane protein ETA_17460 is a protein derived from the bacterium Erwinia tasmaniensis, specifically from the strain DSM 17950/Et1/99 . It belongs to the UPF0756 protein family, a group of uncharacterized proteins with predicted membrane-spanning domains . The designation "UPF" stands for "Uncharacterized Protein Family," indicating that while the protein has been identified and sequenced, its precise biological function remains to be fully elucidated.
The protein is identified in the UniProt database with the accession number B2VET5 and is also known by the identifier Y1746_ERWT9 in some databases . The gene encoding this protein is designated as ETA_17460 in the Erwinia tasmaniensis genome, which explains the protein's common name .
Source Organism
Erwinia tasmaniensis is a gram-negative bacterium belonging to the family Erwiniaceae within the order Enterobacterales . It is a non-pathogenic bacterium in the genus Erwinia, as described in the genome sequencing study by Kube et al. in 2008 . The complete genome of Erwinia tasmaniensis strain Et1/99 has been sequenced, allowing for the identification and characterization of its constituent proteins, including ETA_17460 .
While Erwinia tasmaniensis itself is considered non-pathogenic, other Erwinia-like organisms have been associated with human infections. A case report documented cervical lymphadenitis caused by an Erwinia-like organism that displayed 98.9% 16S rRNA gene sequence similarity to Erwinia tasmaniensis . This suggests potential clinical relevance for understanding proteins from this bacterial genus.
Membrane Topology
According to UniProtKB annotations, ETA_17460 is classified as a cell membrane protein with multiple membrane-spanning domains, making it a multi-pass membrane protein . This subcellular localization was determined through computational prediction methods as indicated by the evidence code "ECO:0000255|HAMAP-Rule:MF_01874" in the UniProt database .
The hydrophobic nature of many segments in the amino acid sequence supports its classification as a membrane protein. These hydrophobic regions likely form transmembrane domains that anchor the protein within the bacterial cell membrane .
Protein Family and Conservation
ETA_17460 belongs to the UPF0756 family, as noted in the UniProtKB entry . This family includes similar proteins found across various bacterial species. The conservation of proteins within this family suggests they may serve important functions in bacterial physiology, despite their current "uncharacterized" status.
The protein has been assigned to the bacterial cluster COG2707 in the eggNOG database, further supporting its evolutionary conservation across bacterial species . This conservation across species suggests a potentially important role in bacterial cell function.
Expression Systems
The recombinant ETA_17460 protein is typically produced using Escherichia coli as the expression host . The full-length protein (amino acids 1-148) is expressed with an N-terminal histidine tag (His-tag), which facilitates protein purification through affinity chromatography . This expression strategy is common for producing bacterial membrane proteins for research purposes.
Reconstitution and Handling
The lyophilized protein should be briefly centrifuged prior to opening to bring the contents to the bottom of the vial . Reconstitution is recommended in deionized sterile water to a concentration of 0.1-1.0 mg/mL . For long-term storage after reconstitution, addition of glycerol to a final concentration of 5-50% is recommended, with 50% being the default concentration suggested by suppliers .
The reconstituted protein should be handled with care to maintain its structural integrity and biological activity. Repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and loss of function .
Research Applications
While the specific research applications for ETA_17460 are not extensively documented in the provided search results, recombinant membrane proteins generally serve several important functions in biological research:
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Structural studies: The purified protein can be used for X-ray crystallography, NMR spectroscopy, or cryo-electron microscopy to determine its three-dimensional structure.
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Antibody production: The recombinant protein can serve as an antigen for generating specific antibodies, which can then be used for immunodetection methods.
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Protein-protein interaction studies: The tagged protein can be used in pull-down assays, co-immunoprecipitation, or yeast two-hybrid screens to identify interaction partners.
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Functional assays: If the protein has enzymatic activity or other measurable functions, the recombinant form can be used in biochemical assays.
The ELISA-compatible form suggests applications in immunological detection and quantification of the protein or in developing diagnostic assays .
Genomic Organization
The ETA_17460 gene was identified during the genome sequencing of Erwinia tasmaniensis strain Et1/99, as reported by Kube et al. in 2008 . This study provided the first complete genome sequence of this non-pathogenic bacterium, enabling the identification and annotation of its constituent genes and proteins.
The genome of Erwinia tasmaniensis strain Et1/99 is deposited in the EMBL database with the accession number CU468135, and the ETA_17460 gene product is registered with the identifier CAO96792.1 . The gene is also referenced in the RefSeq database with the identifier WP_012441481.1 .
Evolutionary Conservation
The ETA_17460 protein shows evolutionary conservation across bacterial species, as evidenced by its inclusion in several comparative genomics databases:
This evolutionary conservation suggests that the protein may serve an important function in bacterial physiology, despite its current "uncharacterized" status. Proteins that are conserved across diverse bacterial species often play fundamental roles in cellular processes.
Predicted Functions Based on Structure
While the specific biological function of ETA_17460 remains unknown, its classification as a multi-pass membrane protein suggests potential roles in:
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Membrane transport: Many multi-pass membrane proteins function as transporters or channels that facilitate the movement of ions, nutrients, or other molecules across the cell membrane.
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Signal transduction: Membrane proteins often serve as receptors or components of signaling pathways that transmit information from the extracellular environment to the cell interior.
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Membrane structure: Some membrane proteins contribute to the structural integrity or organization of the cell membrane.
The Gene Ontology annotation in the UniProtKB entry assigns ETA_17460 to the cellular component term "integral component of plasma membrane" (GO:0005887) , supporting its role as a membrane-embedded protein.
Related Organisms and Clinical Relevance
While Erwinia tasmaniensis itself is considered non-pathogenic, related Erwinia species and Erwinia-like organisms have been associated with human infections. A case report documented cervical lymphadenitis (inflammation of lymph nodes in the neck) caused by an Erwinia-like organism that shared 98.9% 16S rRNA gene sequence similarity with Erwinia tasmaniensis .
In this case, the patient was successfully treated with oral ciprofloxacin for two weeks, leading to complete recovery . The identification of the causative organism was initially made using the Vitek 2 system, which suggested Pantoea species, but subsequent 16S rRNA gene sequence analysis confirmed it as an Erwinia-like organism .
This clinical report highlights the potential relevance of understanding proteins from Erwinia species, as some members of this genus or closely related organisms may be associated with human infections, albeit rarely.
Future Research Prospects
Several avenues for future research on ETA_17460 could be pursued:
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Structural determination: Advanced techniques such as cryo-electron microscopy or X-ray crystallography could reveal the three-dimensional structure of the protein, providing insights into its potential function.
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Protein-protein interaction studies: Identifying the interaction partners of ETA_17460 could help elucidate its role in cellular processes.
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Gene knockout or knockdown studies: Investigating the effects of ETA_17460 deletion or reduced expression on Erwinia tasmaniensis physiology could reveal its importance for bacterial survival or specific functions.
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Comparative studies with homologous proteins: Analyzing similar proteins from related bacterial species could provide evolutionary insights and functional clues.
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Proteomic studies: Mass spectrometry-based approaches could identify post-translational modifications or expression patterns of the protein under various conditions.
Product Specs
Note: We prioritize shipping the format currently in stock. However, if you have specific format requirements, please indicate them during order placement. We will accommodate your request to the best of our ability.
Note: All proteins are shipped standard with normal blue ice packs. If dry ice shipping is required, please communicate with us in advance, as additional charges will apply.
Generally, liquid form maintains a shelf life of 6 months at -20°C/-80°C. Lyophilized form exhibits a shelf life of 12 months at -20°C/-80°C.
Target Background
KEGG: eta:ETA_17460
STRING: 465817.ETA_17460
Q&A
What is Erwinia tasmaniensis UPF0756 membrane protein ETA_17460?
ETA_17460 is a membrane protein belonging to the UPF0756 protein family found in Erwinia tasmaniensis, a non-phytopathogenic bacterium isolated from apple and pear trees in Australia. The full-length protein consists of 148 amino acids and functions as an integral membrane protein. The protein sequence suggests multiple transmembrane domains typical of membrane transport proteins, though its specific physiological role remains under investigation. The recombinant version is commonly expressed with a histidine tag to facilitate purification and experimental applications .
What is the amino acid sequence of ETA_17460?
The full amino acid sequence of the ETA_17460 protein (1-148) is:
MFDLTLAIMLFLAALSYFSHNITVTIALLVLIVIRMTPLQQTFPWIEKQGMTVGIIILTIGVMAPIASGTIPSSTLMHSFLHWKSLTAIAIGIFVSWLGGRGVTLMSTQPTVVGGLLIGTIIGVSLFRGVPVGPLIAAGLLSLMLGKG
What is the taxonomic context of Erwinia tasmaniensis?
Erwinia tasmaniensis belongs to the family Enterobacteriaceae, which contains several genera of clinical and agricultural importance. E. tasmaniensis was first described in 2006 by Geider et al. as a non-phytopathogenic bacterium isolated from apple and pear trees in Australia. It is closely related to other Erwinia species, including E. toletana (98.8% 16S rRNA similarity) and E. billingiae (98.1% similarity). While most Erwinia species are plant-associated or plant pathogens, a related Erwinia-like organism with 98.9% 16S rRNA similarity to E. tasmaniensis has been associated with a human case of cervical lymphadenitis, representing a rare clinical association .
What are the predicted structural features of ETA_17460 protein?
Based on sequence analysis, ETA_17460 displays characteristics typical of integral membrane proteins with multiple predicted transmembrane helices. The 148-amino acid sequence contains predominantly hydrophobic regions interspersed with charged and polar residues at predicted membrane interfaces. Secondary structure prediction suggests an alpha-helical rich structure, consistent with its classification as a membrane protein. The protein contains regions with sequence signatures associated with transporter activity, though crystallographic studies would be necessary to confirm the detailed three-dimensional structure .
How does ETA_17460 compare to homologous proteins in other bacterial species?
ETA_17460 belongs to the UPF0756 protein family, which is conserved across multiple bacterial genera. Homology analysis reveals significant sequence conservation within Erwinia species, with varying degrees of conservation in other Enterobacteriaceae. Proteins with similar sequence and predicted structural features can be found in related genera such as Pantoea, which was initially confused with the Erwinia isolate in clinical identification. Functional characterization of these homologs remains limited, making comparative functional studies an important area for future research .
What expression systems are available for recombinant production of ETA_17460?
Multiple expression systems have been developed for ETA_17460 recombinant production, each with distinct advantages:
| Expression System | Tag Options | Advantages | Considerations |
|---|---|---|---|
| E. coli | His, Avi-tag Biotinylated | High yield, cost-effective, rapid production | Potential improper folding of membrane proteins |
| Yeast | Various tags available | Better for eukaryotic-like post-translational modifications | Longer production time than E. coli |
| Baculovirus | Various tags available | Effective for complex membrane proteins | More complex system, higher cost |
| Mammalian cells | Various tags available | Optimal for complex folding and modifications | Highest cost, longer production time |
The E. coli system with His-tagging represents the most commonly used approach for initial characterization studies, while more complex expression systems may be preferred for detailed structural and functional analyses .
What purification strategies are most effective for recombinant ETA_17460?
Purification of membrane proteins like ETA_17460 requires specialized approaches. The recommended protocol involves:
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Initial solubilization using appropriate detergents (e.g., n-dodecyl-β-D-maltoside or CHAPS)
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Affinity chromatography using the His-tag (immobilized metal affinity chromatography)
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Optional size exclusion chromatography for higher purity
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Buffer optimization to maintain protein stability
For functional studies, it's crucial to maintain the protein in a native-like membrane environment, potentially using nanodiscs, liposomes, or detergent micelles. The protein achieves >90% purity through these methods, as determined by SDS-PAGE analysis .
What are the optimal storage conditions for purified ETA_17460?
For maximum stability and activity retention, recombinant ETA_17460 should be stored according to the following recommendations:
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Long-term storage: Lyophilized powder at -20°C/-80°C
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Working aliquots: 4°C for up to one week
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Reconstitution: Use deionized sterile water to a concentration of 0.1-1.0 mg/mL
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Cryoprotection: Add 5-50% glycerol (final concentration) for freeze-thaw protection
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Storage buffer: Tris/PBS-based buffer, 6% Trehalose, pH 8.0
Repeated freeze-thaw cycles should be avoided to prevent protein degradation and activity loss. Proper aliquoting immediately after reconstitution is essential for maintaining protein integrity over multiple experiments .
How can recombinant ETA_17460 be used in bacterial physiology studies?
Recombinant ETA_17460 serves as a valuable tool for studying bacterial membrane biology and physiology through several experimental approaches:
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Antibody production for localization and expression studies
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Protein-protein interaction analyses to identify functional partners
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Reconstitution in artificial membrane systems to study transport properties
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Comparative studies with homologs from pathogenic Erwinia species
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Structure-function relationship investigations through mutational analysis
These applications can provide insights into bacterial membrane organization, transport mechanisms, and potential roles in bacterial adaptation to environmental conditions .
What techniques are suitable for studying ETA_17460 interactions with other proteins?
Several methodologies are appropriate for investigating protein interactions involving ETA_17460:
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Pull-down assays using the His-tag or other affinity tags
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Bacterial two-hybrid systems adapted for membrane proteins
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Crosslinking studies followed by mass spectrometry
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Förster resonance energy transfer (FRET) with fluorescently labeled proteins
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Surface plasmon resonance for quantitative binding kinetics
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Co-immunoprecipitation with custom antibodies against ETA_17460
When designing these experiments, it's essential to maintain the membrane protein in conditions that preserve its native conformation, typically using mild detergents or membrane mimetics .
How can researchers investigate the potential substrate specificity of ETA_17460?
Determining substrate specificity for uncharacterized membrane proteins like ETA_17460 requires systematic approaches:
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Liposome reconstitution and transport assays with radiolabeled potential substrates
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Electrophysiological measurements in planar lipid bilayers
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In silico docking studies with candidate substrates
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Comparative genomics to identify conserved domains associated with specific substrate classes
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Site-directed mutagenesis of predicted substrate-binding residues followed by functional assays
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Phenotypic analysis of knockout or overexpression strains under various growth conditions
A combination of these approaches typically provides complementary evidence to narrow down potential physiological substrates .
What are common challenges in expressing and purifying ETA_17460, and how can they be addressed?
Membrane proteins like ETA_17460 present several challenges during recombinant expression and purification:
| Challenge | Cause | Solution Strategies |
|---|---|---|
| Low expression yield | Toxicity to host, protein aggregation | Use tightly controlled induction systems, lower temperature expression, specialized E. coli strains (C41/C43) |
| Protein aggregation | Improper folding, hydrophobic interactions | Optimize detergent type and concentration, add stabilizing agents like glycerol or trehalose |
| Low solubility | Hydrophobic transmembrane domains | Screen multiple detergents, consider fusion partners that enhance solubility |
| Loss of native conformation | Harsh purification conditions | Use milder detergents, implement negative purification steps, verify activity after each step |
| Protein instability | Proteolytic degradation | Add protease inhibitors, reduce purification time, optimize buffer conditions |
Systematic optimization of expression conditions and purification protocols is often necessary for each specific application .
How can researchers verify the proper folding and functionality of purified ETA_17460?
Verifying proper folding and functionality of membrane proteins requires multiple complementary approaches:
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Circular dichroism spectroscopy to assess secondary structure content
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Limited proteolysis to probe structural integrity
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Size exclusion chromatography to evaluate monodispersity
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Reconstitution into liposomes followed by functional assays
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Thermal stability assays with fluorescent dyes (e.g., CPM assay)
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Binding assays with known ligands or antibodies specific to correctly folded protein
These methods collectively provide evidence for proper folding and potential functionality, particularly important when working with membrane proteins that are prone to misfolding during recombinant expression .
What controls should be included in experiments involving recombinant ETA_17460?
Rigorous experimental design requires appropriate controls:
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Empty vector controls for expression studies
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Heat-denatured protein controls for functional assays
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Tag-only protein controls to distinguish tag-mediated effects
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Homologous proteins from related species for specificity determination
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Predicted inactive mutants (e.g., mutations in conserved residues)
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Detergent-only controls for membrane protein assays
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Competitive inhibition controls for binding studies
These controls help distinguish specific protein-mediated effects from experimental artifacts, particularly important when working with membrane proteins in artificial environments .
How does ETA_17460 relate to potential virulence factors in pathogenic Erwinia species?
While E. tasmaniensis is non-pathogenic to plants, related Erwinia species are significant plant pathogens. Comparative analysis between ETA_17460 and homologs in pathogenic species may reveal:
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Potential roles in bacterial adaptation to host environments
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Conservation of membrane organization between pathogenic and non-pathogenic species
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Evolutionary divergence patterns correlating with pathogenicity
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Possible involvement in stress response or nutrient acquisition during host colonization
What insights can functional genomics provide about the physiological role of ETA_17460?
Functional genomics approaches can illuminate the physiological context of ETA_17460:
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Transcriptomic analysis to identify co-expressed genes under various conditions
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Analysis of genomic neighborhood for functionally related genes
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Comparative genomics across Erwinia species to identify conserved operons
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Identification of regulatory elements controlling expression
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Metabolomic studies comparing wild-type and knockout strains
Gene expression patterns under different environmental conditions can be particularly informative, potentially revealing conditions where ETA_17460 expression is significantly altered, suggesting physiological importance .
What are the implications of finding Erwinia-like organisms in human clinical samples?
The isolation of an Erwinia-like organism (closely related to E. tasmaniensis) from a human cervical lymphadenitis case raises several research questions:
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Potential pathogenic mechanisms in opportunistic human infections
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Virulence factors shared between plant-associated and clinical isolates
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Antibiotic susceptibility profiles of Erwinia clinical isolates
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Diagnostic challenges in correctly identifying Erwinia in clinical settings
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Evolutionary adaptations enabling colonization of diverse hosts
The clinical isolate showed phenotypic differences from E. tasmaniensis, such as utilizing rhamnose but not citrate, and reducing nitrates to nitrites. These differences highlight the need for detailed characterization of membrane proteins like ETA_17460 that may contribute to adaptation to different environments .
