Recombinant Escherichia coli O8 UPF0756 membrane protein YeaL (yeaL)
Description
Introduction to Recombinant Escherichia coli O8 UPF0756 membrane protein YeaL
The Recombinant Escherichia coli O8 UPF0756 membrane protein YeaL, identified by the gene name yeaL and UniProt ID B7M1K3, represents a significant yet poorly characterized membrane protein in E. coli O8 strains. As a member of the UPF0756 classification, this protein remains functionally enigmatic despite having been identified and sequenced. The "UPF" designation specifically indicates that the protein belongs to a family whose functions have not yet been definitively established through experimental verification .
The recombinant form of YeaL consists of the full-length sequence spanning 148 amino acids, produced with an N-terminal histidine tag that facilitates purification and experimental applications. This His-tagged version is typically expressed in E. coli expression systems, providing researchers with a convenient source for biochemical, structural, and functional studies. The production of recombinant YeaL enables detailed investigation of this membrane protein outside its native context .
Integral membrane proteins like YeaL constitute approximately 20-30% of all proteins in most organisms and perform vital cellular functions, including transport, signaling, and maintenance of membrane integrity. Understanding the structure and function of membrane proteins such as YeaL is particularly challenging due to their hydrophobic nature and difficulties in crystallization for structural determination. Nevertheless, these proteins represent valuable subjects for research due to their essential roles in bacterial physiology and potential as targets for antimicrobial development.
Physical and Chemical Properties
The Recombinant Escherichia coli O8 UPF0756 membrane protein YeaL exhibits several key physical and chemical properties that influence its behavior in experimental settings and potential functional characteristics. As an integral membrane protein, YeaL is inherently hydrophobic, making it challenging to work with under standard aqueous conditions without appropriate detergents or membrane mimetics to maintain solubility and native structure .
The recombinant protein is produced with greater than 90% purity as determined by SDS-PAGE analysis, ensuring reliability for downstream applications and experimental investigations. While the exact molecular weight has not been explicitly specified in the available data, the 148-amino acid sequence with an N-terminal His tag would yield a protein with an approximate molecular weight in the range of 15-17 kDa, depending on the exact length of the histidine tag attached .
Commercial preparations of the recombinant YeaL protein are typically supplied in lyophilized form, which enhances stability during shipping and long-term storage. The recommended storage buffer consists of a Tris/PBS-based formulation containing 6% Trehalose at pH 8.0. This specific buffer composition serves multiple purposes: the Tris/PBS component provides pH stability and physiological salt concentration, while the inclusion of 6% Trehalose acts as a cryoprotectant and stabilizing agent that helps preserve protein structure during freeze-thaw cycles and prevents aggregation .
For reconstitution of the lyophilized protein, it is recommended to dissolve it in deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL. For long-term storage of the reconstituted protein, the addition of glycerol to a final concentration between 5-50% is advised, with 50% being the standard recommendation for optimal preservation at -20°C/-80°C. These specific handling requirements underscore the delicate nature of membrane proteins and the care required to maintain their structural and functional integrity for research applications .
Production and Purification Methods
The production of Recombinant Escherichia coli O8 UPF0756 membrane protein YeaL involves sophisticated recombinant DNA technology and protein expression systems optimized for membrane protein production. The process begins with the molecular cloning of the yeaL gene, encoding amino acids 1-148 of the protein, into a suitable expression vector that incorporates an N-terminal histidine tag sequence. This genetic construct is then transformed into specialized E. coli expression strains that have been optimized for recombinant protein production .
After transformation, bacterial cultures are grown under controlled conditions and protein expression is induced using appropriate induction systems, commonly IPTG (Isopropyl β-D-1-thiogalactopyranoside) for lac promoter-based vectors. Following expression, the bacterial cells are harvested and disrupted to release cellular contents. The membrane fraction containing the recombinant YeaL protein is then isolated through differential centrifugation steps .
The N-terminal histidine tag plays a crucial role in the subsequent purification process by enabling immobilized metal affinity chromatography (IMAC). In this technique, the expressed protein selectively binds to metal ions (typically nickel or cobalt) immobilized on a chromatography matrix, allowing effective separation from the complex mixture of cellular components. Following elution from the IMAC column using imidazole-containing buffers, additional purification steps may include size exclusion chromatography or ion exchange chromatography to achieve the reported purity level exceeding 90% as determined by SDS-PAGE analysis .
For membrane proteins like YeaL, the purification process presents unique challenges due to their hydrophobic nature. Special considerations include the use of appropriate detergents (such as n-Dodecyl β-D-maltoside or Triton X-100) to solubilize the protein from the membrane, careful buffer selection to maintain stability, and potentially the incorporation of lipid mimetics to preserve native-like structure throughout the purification process .
The final purified product is typically prepared in a Tris/PBS-based buffer with 6% Trehalose at pH 8.0 and lyophilized through a controlled freeze-drying process to remove water while preserving the protein's structural integrity. This lyophilization enhances stability for shipping and long-term storage. Quality control measures include SDS-PAGE analysis to confirm purity, protein concentration determination, and potentially functional assays to verify the integrity of the recombinant protein .
Predicted Functions
The specific biological function of the UPF0756 membrane protein YeaL in Escherichia coli remains largely uncharacterized, as indicated by its classification within the "Uncharacterized Protein Family" (UPF) category. Despite this limited functional characterization, several predictions regarding its potential physiological roles can be made based on sequence analysis, membrane localization, and relationships to other proteins .
As an integral membrane protein with multiple predicted transmembrane domains, YeaL likely participates in one or more membrane-associated functions. The hydrophobic nature of its amino acid sequence, with multiple predicted transmembrane segments, suggests possible roles in membrane organization, small molecule transport, or signal transduction across the bacterial cell membrane. The presence of conserved sequence motifs may indicate specific functional domains, though detailed structure-function relationships have yet to be experimentally established .
Genomic context analysis—examining the genes located near yeaL in the E. coli genome—can provide additional clues about potential functions. While limited information is available from the search results, such analyses typically help identify functionally related genes that may operate in the same biochemical pathways or cellular processes. The conservation of this protein across different bacterial species, including variants in Salmonella choleraesuis, suggests it likely performs an important function in bacterial physiology that has been maintained throughout evolutionary history .
Preliminary evidence from studies of other uncharacterized bacterial membrane proteins indicates that many such proteins play roles in stress responses, adaptation to environmental changes, maintenance of membrane potential, or antibiotic resistance. The systematic investigation of previously uncharacterized proteins has frequently revealed unexpected and important functions, suggesting that YeaL may similarly perform a significant role in bacterial cellular processes that has yet to be fully appreciated .
Comparative Analysis with Similar Proteins
Comparative analysis with related proteins provides valuable insights into potential functions through evolutionary relationships. The UPF0756 family includes membrane proteins found in various bacterial species, suggesting a conserved function that has been maintained throughout bacterial evolution .
A close examination of the Salmonella choleraesuis UPF0756 membrane protein YeaL (UniProt ID Q57Q14) reveals high sequence conservation with the E. coli variant, with approximately 95% sequence identity. The differences between these homologs are concentrated in specific regions, potentially reflecting subtle functional adaptations to different cellular environments. Key differences include the substitution of serine (S) with asparagine (N) at position 35, isoleucine (I) with threonine (T) at position 44, and several other minor variations throughout the sequence. These specific amino acid changes might influence substrate specificity if YeaL functions as a transporter, or interaction specificity if it participates in signaling or protein-protein interaction networks .
While not directly related to YeaL, studies of other E. coli membrane proteins of previously unknown function have yielded important insights. For example, YqjA, a member of the DedA/Tvp38 membrane protein family, has been identified as a putative osmosensing transporter required for growth at alkaline pH. This example illustrates how membrane proteins initially classified as "uncharacterized" can later be discovered to perform specific and important functions in bacterial physiology, suggesting similar possibilities for YeaL .
The classification of proteins within UPF families represents an intermediate stage in our understanding of bacterial proteomes. As research progresses, these proteins are frequently reclassified into functional categories once their biological roles are experimentally determined. Integration of genomic, proteomic, and functional data will be essential for definitively establishing the role of YeaL in E. coli physiology .
Applications and Research Significance
Recombinant Escherichia coli O8 UPF0756 membrane protein YeaL offers significant potential for various research applications and scientific investigations. The availability of purified recombinant YeaL provides a valuable tool for researchers studying bacterial membrane biology, protein structure-function relationships, and potentially bacterial physiology under different environmental conditions .
As a purified recombinant protein, YeaL enables several key research applications:
Structural studies represent a primary application area, where the purified protein can be utilized for structural determination through techniques such as X-ray crystallography, cryo-electron microscopy, or NMR spectroscopy. Such structural insights would contribute significantly to our understanding of membrane protein architecture and potentially reveal functional sites within the protein. The high purity (>90%) of the commercially available recombinant protein makes it particularly suitable for these demanding structural biology applications .
Immunological applications constitute another important research area, as the recombinant protein can serve as an immunogen for antibody development. These antibodies can subsequently be employed for protein detection, localization studies using immunofluorescence microscopy, or potentially therapeutic applications if YeaL is found to be accessible on the bacterial cell surface. The His-tagged version facilitates easy detection using anti-His antibodies during preliminary studies .
Functional characterization represents perhaps the most significant potential application. The purified protein can be reconstituted into artificial membrane systems such as liposomes or nanodiscs for in vitro functional assays to elucidate its biochemical activities, potential transport functions, or interactions with other cellular components. Such studies could definitively establish the biological role of this currently uncharacterized protein .
Understanding membrane proteins like YeaL is particularly significant because they constitute approximately one-third of all proteins encoded in bacterial genomes and perform critical functions in cellular processes. Additionally, bacterial membrane proteins frequently serve as targets for antimicrobial development, with a substantial percentage of current antibiotics targeting membrane-associated processes. The study of previously uncharacterized proteins like YeaL contributes to filling knowledge gaps in our understanding of bacterial proteomes and potentially revealing novel aspects of bacterial physiology .
Product Specs
Please note: We will prioritize shipping the format currently in stock. However, if you have a specific format requirement, please indicate it in your order notes. We will prepare the product according to your request.
Note: All our proteins are shipped with standard blue ice packs by default. If dry ice shipping is required, please inform us in advance. 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: ecr:ECIAI1_1853
Q&A
What is the UPF0756 membrane protein YeaL and what are its key characteristics?
The UPF0756 membrane protein YeaL is a membrane-bound protein from Escherichia coli O8 consisting of 148 amino acids (full length). It belongs to the UPF0756 protein family which is still being characterized for specific biological functions. The protein has the UniProt ID B7M1K3 and is typically produced as a recombinant protein with an N-terminal His tag for purification purposes .
Key characteristics include:
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Full amino acid sequence: MFDVTLLILLGLAALGFISHNTTVAVSILVLIIVRVTPLSTFFPWIEKQGLSIGIIILTIGVMAPIASGTLPPSTLIHSFLNWKSLVAIAVGVIVSWLGGRGVTLMGSQPQLVAGLLVGTVLGVALFRGVPVGPLIAAGLVSLIVGKQ
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Predominantly hydrophobic amino acid composition, consistent with its membrane localization
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Likely contains multiple transmembrane domains based on its sequence
How is recombinant YeaL protein typically expressed and purified?
Recombinant YeaL protein is typically expressed in E. coli expression systems. The standard methodology involves:
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Cloning the yeaL gene into an expression vector with an N-terminal His tag
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Transforming the construct into a suitable E. coli expression strain
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Inducing protein expression under optimized conditions
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Lysing cells and purifying the protein through affinity chromatography using the His tag
The purified protein is often obtained as a lyophilized powder with purity greater than 90% as determined by SDS-PAGE. For storage and handling, it's recommended to:
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Store at -20°C/-80°C upon receipt
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Aliquot to avoid repeated freeze-thaw cycles
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Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL
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Add 5-50% glycerol (final concentration) for long-term storage
What expression vectors are most suitable for YeaL protein production?
When selecting expression vectors for YeaL protein production, several factors must be considered:
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Promoter strength: Strong promoters like T7 are typically used for membrane proteins but may require fine-tuning to prevent inclusion body formation
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Affinity tags: N-terminal His tags are commonly used for YeaL purification
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Selection markers: While ampicillin is commonly used, it should be noted that β-lactamase secretion can lead to antibiotic depletion. More stable alternatives like tetracycline may provide better selective pressure during extended cultivation
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Copy number: Medium to low copy number plasmids often provide better expression for membrane proteins by preventing cellular toxicity
For YeaL specifically, vectors providing moderately controlled expression with N-terminal His tags have proven effective in laboratory settings .
What are the optimal conditions for maximizing soluble YeaL protein expression?
Maximizing soluble YeaL protein expression requires optimization of several parameters:
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Temperature modulation: Lower temperatures (16-25°C) after induction can improve folding and reduce inclusion body formation
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Inducer concentration: Titrating inducer concentration (e.g., IPTG) can help find the optimal expression level that balances yield with proper folding
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N-terminal sequence optimization: Recent research demonstrates that modifying the nucleotides immediately following the start codon can significantly enhance protein expression in a construct-specific manner
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Media composition: For membrane proteins like YeaL, enriched media formulations with additional phospholipids may improve proper membrane insertion
A directed evolution approach using FACS-based selection of N-terminal coding DNA libraries has shown up to 30-fold increases in soluble protein yield for challenging proteins . This methodology could be applied to YeaL protein to optimize expression:
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Create a YeaL-GFP fusion construct
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Generate libraries with varied N-terminal sequences
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Use FACS to select high-expressing variants
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Validate improved expression constructs individually
What are the challenges in structural characterization of YeaL and how can they be addressed?
Structural characterization of membrane proteins like YeaL presents several challenges:
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Maintaining native environment: Traditional structural biology methods often require removing membrane proteins from their native lipid environment, potentially altering their structure and function
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Resolution limitations: Light microscopy techniques lack sufficient resolution to distinguish between truly interacting proteins and those merely adjacent on membranes
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Expression level challenges: Membrane proteins often have expression levels that are either too low or too high for conventional structural studies
Modern approaches to address these challenges include:
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Cryo-electron microscopy (cryo-EM): Recent improvements in cryo-EM have enabled high-resolution imaging of membrane proteins in near-native states
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Native-nanoBleach microscopy: This newer method maintains the native membrane environment while studying protein organization
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Conformational analysis: Capturing different conformational states to produce "3D movies" of the protein's working cycle
For YeaL specifically, a combined approach using both structural (cryo-EM) and functional assays would likely provide the most comprehensive characterization.
How can isotope labeling be incorporated for NMR studies of YeaL?
For NMR studies of YeaL, incorporating isotope labeling requires a systematic approach:
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Expression system selection: Use E. coli strains optimized for membrane protein expression (e.g., C41(DE3), C43(DE3))
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Minimal media preparation: Formulate minimal media with 15N-labeled ammonium chloride and/or 13C-labeled glucose as the sole nitrogen and carbon sources
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Growth protocol:
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Grow cells in rich media to mid-log phase
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Wash and transfer to isotope-enriched minimal media
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Allow adaptation period (30-60 min)
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Induce protein expression at reduced temperature (16-20°C)
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Optimization of induction: Use lower inducer concentrations and longer expression times (16-24 hours) to maximize proper folding
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Detergent screening: Test various detergents for optimal solubilization while maintaining protein structure
This approach allows for the incorporation of NMR-visible isotopes into the protein structure while maintaining proper folding and membrane insertion.
What is known about the structure-function relationship of YeaL and similar UPF0756 family proteins?
The structure-function relationship of YeaL and UPF0756 family proteins remains largely uncharacterized, presenting an opportunity for novel research. Based on sequence analysis and general membrane protein principles:
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Predicted structure: The amino acid sequence (MFDVTLLILLGLAALGFISHNTTVAVSILVLIIVRVTPLSTFFPWIEKQGLSIGIIILTIGVMAPIASGTLPPSTLIHSFLNWKSLVAIAVGVIVSWLGGRGVTLMGSQPQLVAGLLVGTVLGVALFRGVPVGPLIAAGLVSLIVGKQ) suggests multiple transmembrane domains with predominantly hydrophobic character
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Functional hypotheses: While specific functions remain undetermined, the membrane localization suggests possible roles in:
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Small molecule or ion transport
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Signal transduction
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Membrane integrity maintenance
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Protein-protein interactions within membrane complexes
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Advanced research approaches should focus on:
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Generating high-resolution structures using cryo-EM techniques that have proven successful for other membrane proteins
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Identifying potential interaction partners through co-immunoprecipitation or proximity labeling techniques
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Comparative analysis with other UPF0756 family members across bacterial species
How can site-directed mutagenesis be employed to identify critical functional residues in YeaL?
A systematic site-directed mutagenesis approach for identifying critical YeaL residues would include:
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Computational analysis: Begin with in silico prediction of:
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Conserved residues across UPF0756 family proteins
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Predicted transmembrane domains and topological features
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Potential functional motifs or binding sites
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Strategic mutation design:
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Alanine scanning of conserved residues
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Conservative vs. non-conservative substitutions
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Charges-to-alanine mutations in potential binding sites
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Creation of chimeric proteins with related membrane proteins
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Functional assays:
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Membrane localization assessment
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Protein stability and folding analysis
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Growth phenotype analysis in yeaL knockout strains
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Liposome reconstitution for transport activity (if applicable)
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Data interpretation matrix:
| Mutation Category | Expected Outcome if Critical | Experimental Readout |
|---|---|---|
| Conserved residues | Disrupted function/localization | Growth defects, mislocalization |
| Transmembrane domains | Misfolding, aggregation | Reduced expression, inclusion bodies |
| Charged residues | Altered interaction profile | Changed binding partners, activity |
| Terminal regions | Modified processing/targeting | Cellular distribution changes |
What approaches can be used to investigate YeaL protein interactions within the membrane environment?
Investigating YeaL protein interactions within native membrane environments requires specialized techniques:
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In vivo crosslinking:
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Photo-activatable or chemical crosslinkers incorporated at specific positions
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UV-triggered capture of transient interactions
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MS/MS analysis of crosslinked peptides for partner identification
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Proximity labeling:
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Fusion of YeaL with enzymes like BioID or APEX2
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Temporal control of labeling to capture dynamic interactions
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Streptavidin pulldown and mass spectrometry analysis
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Native-nanoBleach microscopy approach:
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Complementary genetic approaches:
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Synthetic genetic array analysis to identify genetic interactions
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Suppressor screening to identify functional relationships
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Two-hybrid membrane protein interaction systems
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These methods collectively provide a comprehensive view of YeaL's interaction network while preserving the critical membrane context.
How can aggregation and inclusion body formation be minimized during YeaL expression?
Minimizing aggregation and inclusion body formation for YeaL requires a multi-faceted approach:
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Expression condition optimization:
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Reduce induction temperature to 16-20°C
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Lower inducer concentration (IPTG) to 0.1-0.5 mM
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Use slower expression rates with weaker promoters or lower copy number plasmids
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Co-expression strategies:
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Molecular chaperones (GroEL/GroES, DnaK/DnaJ)
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Membrane protein-specific folding factors
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Fusion partners known to enhance solubility (MBP, TrxA, GST)
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Media and buffer optimization:
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Supplement with osmolytes like glycerol, trehalose, or arginine
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Increase membrane fluidity with phospholipid supplementation
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Optimize salt concentration and pH for stability
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N-terminal sequence engineering:
What strategies can address problems in YeaL protein purification and stability?
Common challenges in YeaL purification and stability can be addressed through:
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Detergent optimization matrix:
| Detergent Type | Advantages | Limitations | Best For |
|---|---|---|---|
| Mild (DDM, LMNG) | Maintains structure | Less efficient extraction | Functional studies |
| Zwitterionic (CHAPS) | Good solubilization | Potential denaturation | Initial screening |
| Nonionic (Triton X-100) | Efficient extraction | May disrupt some interactions | Mass extraction |
| Styrene Maleic Acid (SMA) | Native lipid environment | Limited compatibility | Native state studies |
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Buffer optimization:
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Chromatography strategies:
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Two-step purification: IMAC followed by size exclusion
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Gradient elution to separate differentially folded species
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On-column refolding protocols for recovering from inclusion bodies
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Stability assessment:
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Thermal shift assays to identify stabilizing conditions
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Limited proteolysis to identify flexible/unstable regions
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Long-term storage stability testing at different temperatures
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How can functional assays be developed for a protein like YeaL with unknown function?
Developing functional assays for YeaL requires a systematic discovery approach:
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Comparative genomics and bioinformatics:
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Analyze gene neighborhood and co-occurrence patterns
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Identify proteins with similar structural predictions
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Search for conserved motifs suggestive of specific functions
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Phenotypic characterization:
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Generate clean gene deletions and assess phenotypes under various conditions
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Perform growth curve analysis under different stressors
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Assess membrane integrity and composition changes
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Reconstitution studies:
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Incorporate purified YeaL into liposomes or nanodiscs
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Monitor changes in membrane properties (fluidity, permeability)
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Test for transport activity of various substrates
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High-throughput screening approaches:
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Metabolite profiling of knockout vs. wild-type cells
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Transcriptome analysis to identify affected pathways
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Chemical genetic screening for compound sensitivity
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Structural biology integration:
How might recent advances in membrane protein structural biology apply to YeaL research?
Recent advances in membrane protein structural biology offer exciting new possibilities for YeaL research:
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Cryo-EM technological improvements:
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Native environment preservation:
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Conformational dynamics visualization:
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Integration with computational approaches:
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Molecular dynamics simulations incorporating lipid bilayers
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AI-assisted structure prediction specifically trained on membrane proteins
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Virtual screening for potential interacting molecules or drugs
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What are the potential physiological roles of YeaL based on current understanding of similar membrane proteins?
While the specific function of YeaL remains to be fully characterized, several hypotheses can be formulated based on its features and similarities to other membrane proteins:
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Transport functions:
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Signaling pathway involvement:
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Cellular homeostasis:
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Membrane proteins regulate metabolic processes
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YeaL may participate in maintaining cellular balance of specific molecules
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Its conservation suggests an important physiological role
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Stress response:
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Many uncharacterized membrane proteins are involved in bacterial stress responses
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YeaL might be activated under specific environmental conditions
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This could explain why its function remains elusive under standard laboratory conditions
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How can systems biology approaches contribute to understanding YeaL function in the broader context of bacterial physiology?
Systems biology offers powerful tools to contextualize YeaL within bacterial physiology:
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Multi-omics integration:
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Combine transcriptomics, proteomics, and metabolomics data from YeaL knockout strains
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Create network models of affected pathways
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Identify condition-specific roles that may not be apparent under standard conditions
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Protein-protein interaction networks:
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Map YeaL's interaction partners through proximity labeling and co-immunoprecipitation
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Analyze the functional categories of interacting proteins
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Identify protein complexes that include YeaL
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Comparative genomics across bacterial species:
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Analyze conservation patterns and genetic context
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Identify co-evolution with other genes suggesting functional relationships
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Examine distribution across bacterial lineages to infer evolutionary importance
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Synthetic biology approaches:
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Create synthetic circuits incorporating YeaL to test functional hypotheses
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Engineer reporter systems linked to potential YeaL activities
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Design minimal systems to isolate and characterize YeaL function
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Environmental and stress response mapping:
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Profile YeaL expression and activity across diverse growth conditions
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Identify specific stressors that alter YeaL behavior
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Map condition-specific protein interactions and modifications
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