Recombinant Escherichia coli UPF0756 membrane protein YeaL (yeaL)
Description
Protein Identification and Nomenclature
UPF0756 membrane protein YeaL represents one of the numerous uncharacterized protein families (UPF) found in the E. coli genome. The protein is encoded by the yeaL gene and has been assigned several identifiers in various databases, including UniProtKB accession numbers P0ACY7 (for E. coli CFT073 strain) and C5W4W1 . The designation "UPF0756" indicates its classification in protein family 0756 of functionally uncharacterized proteins in the UniProt database. The protein has several synonyms in the literature, including "yeaL; ECBD_1856; ECD_01758; B21_01746; UPF0756 membrane protein YeaL" .
The protein is categorized as a membrane protein based on computational predictions and sequence analysis, which indicate the presence of multiple transmembrane segments. This classification aligns with the hydrophobic nature of significant portions of its amino acid sequence, characteristic of proteins that integrate into lipid bilayers.
Genomic and Evolutionary Context
YeaL appears to be conserved among different strains of E. coli, including laboratory strains and pathogenic variants such as E. coli CFT073 (a uropathogenic strain) . This conservation across various E. coli lineages suggests the protein may serve an important function in bacterial physiology, despite our current limited understanding of its specific role.
Predicted Three-Dimensional Structure
While no experimentally determined structure of YeaL is available in the search results, computational modeling approaches have been applied to predict its three-dimensional conformation. Notably, AlphaFold has generated a structural model (AF-P0ACY7-F1) for YeaL from E. coli CFT073 . This model was released to the AlphaFold Database on December 9, 2021, with the last modification date of September 30, 2022.
Membrane Topology and Integration
Based on its sequence characteristics and computational predictions, YeaL is expected to adopt a multi-pass transmembrane topology. The hydrophobic stretches within its sequence likely form alpha-helical segments that span the bacterial cytoplasmic membrane. These transmembrane domains would anchor the protein within the lipid bilayer, while connecting loop regions would extend into either the cytoplasm or periplasm of the bacterial cell.
Expression and Purification Strategies
Recombinant YeaL protein has been successfully produced in E. coli expression systems. Commercial sources offer the full-length protein (amino acids 1-148) fused to an N-terminal histidine tag . This approach facilitates purification using affinity chromatography techniques that leverage the specific interaction between the histidine tag and metal ions.
The protein is typically expressed in E. coli, which represents both the source organism for the native gene and the expression host for recombinant production. This homologous expression system may offer advantages for proper folding and membrane integration of this bacterial membrane protein, although specific details on expression optimization are not provided in the search results.
Table 1: Recombinant YeaL Protein Specifications
Research Applications
While specific research applications of recombinant YeaL are not explicitly described in the search results, as an uncharacterized membrane protein, it presents several potential uses in both basic and applied research:
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Structural studies: The availability of purified recombinant protein enables structural characterization through techniques such as X-ray crystallography, cryo-electron microscopy, or nuclear magnetic resonance spectroscopy, which could provide insights beyond computational predictions.
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Functional characterization: Purified YeaL could be employed in biochemical assays to investigate potential enzymatic activities, ligand-binding properties, or protein-protein interactions.
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Antibody production: The recombinant protein could serve as an antigen for generating antibodies, which would facilitate immunodetection of native YeaL in cellular contexts.
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Comparative studies: As a member of the UPF0756 family, YeaL could be used in comparative analyses with related proteins from other bacteria to explore evolutionary relationships and potential conserved functions.
Membrane Protein Families and Analogous Systems
Given the limited information on YeaL's function, insights might be gained by examining analogous membrane proteins in E. coli. While not directly related to YeaL, another E. coli membrane protein, RseP (formerly called YaeL), provides an example of how membrane proteins can play crucial roles in cellular processes. RseP functions as a regulated intramembrane proteolysis (RIP) protease that cleaves within transmembrane sequences of certain proteins .
RseP contains zinc metalloprotease active-site motifs and four transmembrane segments and is essential for cell viability through its role in activating the σE pathway of extracytoplasmic stress responses . While there is no evidence in the search results suggesting YeaL has similar proteolytic activity, this example illustrates how bacterial membrane proteins can serve specialized functions critical to cellular physiology.
Current Limitations in YeaL Research
Despite the availability of recombinant YeaL protein and computational structural models, significant gaps remain in our understanding of this protein:
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Functional characterization: The specific biological role of YeaL in E. coli physiology remains unknown. Comprehensive functional studies, including gene knockout/knockdown approaches, protein interaction analyses, and activity assays would provide valuable insights.
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Experimental structure determination: While computational models offer predictions, experimental determination of YeaL's structure would provide more definitive structural information, potentially revealing functional sites.
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Expression patterns and regulation: Information about when and under what conditions yeaL is expressed, as well as the regulatory mechanisms controlling its expression, could provide contextual clues about its function.
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Evolutionary conservation: More extensive analysis of YeaL homologs across bacterial species beyond E. coli might reveal conserved functional domains or evolutionary patterns.
Emerging Methods for Membrane Protein Research
Advancements in membrane protein research methodologies could accelerate our understanding of YeaL:
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Cryo-electron microscopy: This technique has revolutionized structural studies of membrane proteins by eliminating the need for crystallization, which is often challenging for such proteins.
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Mass spectrometry-based approaches: Techniques like hydrogen-deuterium exchange mass spectrometry, chemical cross-linking, and native mass spectrometry can provide insights into protein dynamics, interactions, and conformational states.
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Computational prediction tools: Continuing improvements in algorithms for predicting protein function from sequence and structure could offer new hypotheses about YeaL's role.
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High-throughput functional screening: Systematic approaches to test potential substrates, interaction partners, or conditions affecting YeaL activity could uncover functional clues.
Product Specs
Note: We prioritize shipping the format readily available in our inventory. However, for specific format requirements, please indicate your preference in the order notes. We will accommodate your needs to the best of our ability.
Note: All our proteins are shipped with standard blue ice packs unless otherwise specified. Should you require dry ice shipping, please inform us in advance as additional charges will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. For the lyophilized form, the shelf life is 12 months at -20°C/-80°C.
Target Background
KEGG: ecj:JW1778
STRING: 316385.ECDH10B_1927
Q&A
What expression systems are most effective for recombinant YeaL protein production?
For optimal expression of recombinant YeaL membrane protein, E. coli-based expression systems offer several advantages including rapid growth, high yield, and economic production. The selection of appropriate host strains is critical, as is the optimization of plasmid copy numbers, promoter selection, and codon usage to enhance yields of this eukaryotic protein . High-cell-density bacterial expression methods, including autoinduction and IPTG-induction, can achieve cell densities with OD600 values of 10-20 in standard laboratory conditions using regular incubator shakers without requiring fermentors .
Implementation protocol:
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Select an E. coli strain optimized for membrane protein expression
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Design expression constructs with appropriate promoters (e.g., T7)
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Consider using autoinduction media to reach high cell densities
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Maintain culture in Tris-based buffer with 50% glycerol as used in commercial preparations
How should researchers store and handle YeaL protein preparations?
Recombinant YeaL protein requires careful storage conditions to maintain stability and functionality. The protein should be stored in a Tris-based buffer with 50% glycerol optimized specifically for this protein . For short-term use, working aliquots can be maintained at 4°C for up to one week, while long-term storage requires temperatures of -20°C or -80°C . Repeated freeze-thaw cycles should be avoided as they may compromise protein integrity and functionality .
What is the structural basis for YeaL function in cellular membranes?
YeaL is a small membrane protein consisting of 37 amino acids with a highly hydrophobic composition (MFDVTLLILLGLAALGFISHNTTVAASILVLIIVRV) . This amino acid sequence suggests a transmembrane structure that likely spans the bacterial membrane once. The protein belongs to the UPF0756 family, a group of uncharacterized proteins with predicted membrane localization. The presence of multiple hydrophobic residues (leucine, isoleucine, valine, phenylalanine) reinforces its membrane-associated nature, while the limited presence of charged residues supports its role as an integral membrane protein.
What strategies can overcome challenges in membrane protein YeaL purification?
Purification of recombinant YeaL requires specialized approaches due to its hydrophobic nature and membrane localization. Researchers should consider:
The tag type will be determined during the production process, which affects the specific purification strategy . Researchers should optimize detergent concentrations to prevent protein aggregation while maintaining sufficient amounts of detergent to keep membrane proteins in solution.
How can researchers optimize high-yield expression of YeaL protein in laboratory settings?
To achieve high yields of recombinant YeaL protein, researchers should implement protocols that focus on optimizing bacterial growth conditions and protein expression:
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Medium optimization: Use enriched media formulations that support high cell densities
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Temperature modulation: Lower induction temperatures (16-25°C) can improve proper folding of membrane proteins
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Expression kinetics: Monitor protein expression over time to determine optimal harvest point
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Induction strategy: Consider auto-induction methods which can yield 14-25 mg of NMR triple-labeled proteins and 17-34 mg of unlabeled proteins from a 50-mL culture
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Scale-up considerations: Maintain similar optimization parameters when scaling to larger volumes
Importantly, optimization techniques should address host strain selection, plasmid copy numbers, promoter selection, mRNA stability, and codon usage to significantly enhance the yields of this bacterial membrane protein .
What techniques are most effective for determining YeaL membrane protein topology?
Determining the precise topology of YeaL within the membrane is critical for understanding its function. Researchers should consider multiple complementary approaches:
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Computational prediction: Use algorithms designed for transmembrane protein topology prediction
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Cysteine scanning mutagenesis: Introduce cysteine residues at various positions and assess accessibility
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Fluorescence-based approaches: Employ GFP fusion constructs to determine orientation
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Protease protection assays: Use selective proteolysis to identify exposed regions
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Antibody accessibility studies: Generate antibodies against specific domains to determine orientation
These methods should be used in combination to generate a comprehensive understanding of YeaL's membrane orientation and topology.
How can researchers determine potential interaction partners of YeaL?
Understanding the protein-protein interaction network of YeaL is essential for elucidating its function. Researchers should employ:
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Bacterial two-hybrid systems: Adapted for membrane protein interactions
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Co-immunoprecipitation: Using tagged versions of YeaL to pull down interacting partners
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Cross-linking studies: Chemical cross-linking followed by mass spectrometry
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Proximity labeling approaches: BioID or APEX2 fusion proteins to identify neighboring proteins
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Genetic screening: Suppressor or synthetic lethal screens to identify functional interactions
Careful validation of potential interactions is essential, as membrane protein interactions are prone to artifacts in many standard interaction detection methods.
How conserved is YeaL across different E. coli strains and related bacteria?
YeaL belongs to the UPF0756 family of uncharacterized proteins and appears in various E. coli strains. The specific sequence referenced is from E. coli strain 55989/EAEC . Considering the ongoing E. coli long-term evolution experiment (LTEE) has documented significant phenotypic and genotypic changes across bacterial populations , researchers should conduct comparative genomic analyses to:
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Assess sequence conservation across different E. coli lineages, particularly the major lineages ST-131, ST-73, ST-95, ST-127, and ST-1193 from phylogroup B2, and ST-69 from phylogroup D
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Examine protein presence/absence patterns in related Enterobacteriaceae
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Identify any selective pressures acting on the gene
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Determine whether horizontal gene transfer has contributed to its distribution
Understanding the evolutionary context of YeaL may provide insights into its functional significance in bacterial physiology.
What is known about YeaL expression regulation in different growth conditions?
The regulation of YeaL expression under different environmental conditions remains an active area of research. Systematic approaches should include:
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Transcriptional profiling: RNA-seq analysis across various growth conditions
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Promoter analysis: Characterization of upstream regulatory elements
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Stress response studies: Examination of expression during various cellular stresses
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Growth phase-dependent expression: Analysis across bacterial growth curve
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Reporter gene fusions: Construction of transcriptional and translational fusions to monitor regulation
These approaches can provide insights into the physiological conditions where YeaL function may be most relevant.
How might YeaL be employed in bacterial-based therapeutic delivery systems?
While YeaL's specific function remains to be fully characterized, recombinant E. coli expressing modified membrane proteins have shown potential in therapeutic applications. For example, E. coli expressing invasin can selectively target Peyer's patches for vaccine delivery . Research exploring YeaL's potential in therapeutic applications should consider:
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Possible fusion of YeaL with bioactive peptides or antigens
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Assessment of its role in bacterial adhesion or invasion
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Potential for targeting specific cell types or tissues
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Integration into existing bacterial delivery platforms
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Safety and immunogenicity considerations
These explorations should build upon established methodologies for using recombinant E. coli in therapeutic applications, such as those demonstrated with invasin-expressing E. coli that showed efficacy as an oral vaccine for cancer immunotherapy .
What advanced biophysical methods can elucidate YeaL structure-function relationships?
Due to its small size and hydrophobic nature, YeaL presents both challenges and opportunities for structural characterization:
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NMR spectroscopy: Particularly suitable for small membrane proteins, potentially using the high-yield expression methods that can generate 14-25 mg of NMR triple-labeled proteins from a 50-mL culture
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Cryo-electron microscopy: May require incorporation into larger assemblies or nanodiscs
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X-ray crystallography: Requiring optimization of crystallization conditions for membrane proteins
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Molecular dynamics simulations: To model membrane interactions and conformational changes
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Site-directed spin labeling combined with EPR: To assess dynamics and conformational states
Integration of multiple biophysical approaches will provide the most comprehensive structural understanding of this small membrane protein.
How can researchers address protein aggregation during YeaL expression and purification?
Membrane protein aggregation represents a significant challenge when working with YeaL:
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Expression temperature optimization: Lower temperatures (16-20°C) often reduce aggregation
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Detergent screening: Systematic evaluation of different detergent types and concentrations
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Fusion partner approaches: Addition of solubility-enhancing fusion proteins
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Co-expression with chaperones: Inclusion of molecular chaperones to assist proper folding
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Buffer optimization: Screening different buffer compositions, pH values, and ionic strengths
Implementing a systematic approach to these parameters while monitoring protein solubility and functionality is essential for overcoming aggregation challenges.
What quality control measures should be implemented when working with recombinant YeaL?
Ensuring the quality and consistency of recombinant YeaL preparations is critical for reproducible research:
| Quality Parameter | Assessment Method | Acceptance Criteria |
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| Purity | SDS-PAGE, SEC | >95% pure by densitometry |
| Identity | Mass spectrometry, Western blot | Matching expected mass, immunoreactivity |
| Homogeneity | DLS, Native PAGE | Monodisperse population |
| Stability | Thermal shift assays, time-course activity | Consistent melting temperature, activity retention |
| Functionality | Application-specific assays | Reproducible activity metrics |
| Secondary structure | Circular dichroism | Consistent with predicted α-helical content |
These quality control measures will help ensure that experimental outcomes are attributable to the protein of interest rather than contaminants or degradation products.
