Recombinant Uncharacterized protein YjeT is a small protein of 65 amino acids native to Escherichia coli. The protein is categorized as "uncharacterized" because its specific biological function has not yet been definitively established. Despite this designation, YjeT has been successfully produced in recombinant form with modifications such as histidine tagging to facilitate purification and research applications. The recombinant form maintains the full-length sequence of the native protein while incorporating elements that enhance its utility in laboratory settings.
The recombinant form of YjeT protein is produced using Escherichia coli expression systems with an N-terminal histidine tag to facilitate purification and downstream applications. The following table summarizes the key specifications of recombinant YjeT protein as documented in product information:
Table 1: Specifications of Recombinant YjeT Protein
Parameter | Specification |
---|---|
Organism | Escherichia coli |
Expression System | Escherichia coli |
Fusion Tag | N-terminal His tag |
Protein Length | Full Length (amino acids 1-65) |
Physical Form | Lyophilized powder |
Purity Level | Greater than 90% (SDS-PAGE verified) |
UniProt Identifier | P0AF74 |
Gene Synonyms | YjeT; Z5783; ECs5152 |
Amino Acid Sequence | MNSTIWLALALVLVLEGLGPMLYPKAWKKMISAMTNLPDNILRRFGGGLVVAGVVVYYMLRKTIG |
This information, derived from product specifications provided by Creative Biomart, outlines the key characteristics of commercially available recombinant YjeT protein .
For laboratory applications, the lyophilized recombinant YjeT protein requires proper reconstitution. The following protocol is recommended based on product specifications:
Table 3: Step-by-Step Reconstitution Protocol for YjeT Protein
Step | Procedure |
---|---|
1 | Centrifuge the vial briefly prior to opening |
2 | Reconstitute in deionized sterile water to 0.1-1.0 mg/mL |
3 | For extended storage, add glycerol to 5-50% final concentration |
4 | Prepare aliquots to minimize freeze-thaw cycles |
5 | Store reconstituted aliquots at -20°C to -80°C |
This reconstitution protocol ensures optimal recovery and stability of the recombinant YjeT protein for experimental applications . The specific recommendations for centrifugation prior to opening and the addition of glycerol for long-term storage reflect standard practices for handling sensitive recombinant proteins.
The specific biological function of YjeT protein remains largely uncharacterized according to the available scientific literature. Unlike some other bacterial proteins that have transitioned from "uncharacterized" to functionally defined entities, YjeT's precise role in bacterial physiology is yet to be fully elucidated.
The limited information about YjeT stands in contrast to other formerly uncharacterized bacterial proteins that have been successfully characterized. For example, another E. coli protein initially designated as uncharacterized, YfjG (now renamed RatA), has been identified as a toxin that inhibits 70S ribosome association, effectively blocking the translation initiation step in protein synthesis . This example illustrates the potential significance of currently uncharacterized proteins like YjeT, which may eventually be found to play important roles in fundamental cellular processes.
The availability of purified recombinant YjeT protein enables numerous research applications aimed at determining its structure and function. While the search results do not provide specific examples of YjeT research applications, its recombinant production with a histidine tag suggests several standard approaches that could be applied:
Table 4: Potential Research Applications for Recombinant YjeT
Research Approach | Potential Application |
---|---|
Structural Analysis | Determination of three-dimensional structure through X-ray crystallography or NMR spectroscopy |
Functional Assays | Biochemical tests to identify potential enzymatic activities or cellular functions |
Protein-Protein Interaction Studies | Identification of binding partners to infer functional roles |
Localization Studies | Determination of subcellular localization to suggest functional context |
Comparative Analysis | Comparison with homologous proteins in other bacterial species |
These research approaches represent standard methodologies for characterizing proteins of unknown function and could be applied to YjeT to advance our understanding of its biological role.
While direct functional information about YjeT is limited, research on other bacterial proteins provides valuable context for understanding the potential significance of YjeT. For example, the YjeE protein (distinct from YjeT) has been studied as an essential E. coli protein with ATPase activity that is vital for bacterial viability . Although YjeE is different from YjeT, the methodology employed in its characterization—including site-directed mutagenesis of conserved motifs and in vitro biochemical assays—exemplifies approaches that could be valuable for YjeT characterization.
Similarly, the identification of RatA (formerly YfjG) as a toxin that inhibits 70S ribosome association demonstrates how uncharacterized proteins can eventually be found to have significant roles in fundamental cellular processes . In this case, researchers demonstrated that RatA specifically blocks the formation of 70S ribosomes by binding to 50S subunits, effectively inhibiting the translation initiation step in protein synthesis. This example highlights how detailed biochemical characterization can reveal the functions of previously uncharacterized proteins.
Based on the limited current understanding of YjeT, several research directions would be valuable for advancing knowledge about this protein:
Comprehensive functional characterization through genetic approaches, including gene deletion and complementation studies to determine essentiality and phenotypic effects
Structural determination through advanced techniques to identify potential functional domains and binding sites
Proteomic analyses to identify potential interaction partners and protein complexes involving YjeT
Comparative genomic studies to understand evolutionary conservation and potential functional importance across bacterial species
Expression analysis under various growth conditions to identify regulatory patterns that might suggest functional contexts
These research directions represent logical next steps in the characterization of YjeT and would contribute to filling the significant knowledge gaps that currently exist regarding this protein.
KEGG: sfl:SF4331
YjeT is an uncharacterized bacterial transmembrane protein found in organisms like Escherichia coli. While its specific function remains largely unknown, it belongs to a family of proteins that may play important roles in bacterial physiology. Studying uncharacterized proteins like yjeT is significant because they represent knowledge gaps in our understanding of bacterial proteomes, potentially revealing new biological functions and pathways . Research on such proteins follows the tradition of investigating proteins like YjeQ, which was initially uncharacterized but later found to be indispensable for bacterial growth and possessing unique structural features .
E. coli and yeast expression systems typically offer the best yields and shorter turnaround times for recombinant yjeT production. For applications requiring post-translational modifications or proper protein folding, insect cells with baculovirus or mammalian expression systems may be more appropriate, though with longer production times and potentially lower yields . The selection of expression system should be guided by your specific research requirements, including whether native conformation is essential for functional studies.
A DoE approach can significantly optimize yjeT protein purification by systematically exploring multiple variables simultaneously. This method involves:
Defining objectives and factors (e.g., pH, temperature, salt concentration)
Setting factor ranges (upper and lower limits)
Designing experiments to cover the experimental space efficiently
Analyzing multiple responses (yield, purity, activity)
For example, a DoE study might reveal that specific combinations of heating temperature and additive concentration maximize yield while maintaining protein activity. This approach is more efficient than traditional one-factor-at-a-time methods and can identify unexpected interactions between variables .
Factor | Low Level | High Level | Primary Effect on Yield |
---|---|---|---|
pH | 6.0 | 8.0 | Moderate |
Temperature | 4°C | 30°C | High |
Salt concentration | 0.1M | 0.5M | Moderate |
Additive concentration | 0% | 0.5% | High |
Based on the properties of similar membrane proteins, an effective purification strategy for recombinant yjeT would include:
Cell lysis under conditions that maintain protein stability
Solubilization with appropriate detergents (due to its transmembrane nature)
Initial capture using affinity chromatography (if a tag is included in the recombinant construct)
Further purification via ion exchange chromatography
Final polishing using size exclusion chromatography if higher purity is required
The strategy should be optimized using DoE principles to identify the most critical factors affecting yield and purity. Additionally, the protocol should avoid steps that are challenging to scale up if future applications might require larger quantities .
Verification of yjeT protein purity and integrity should employ multiple complementary techniques:
SDS-PAGE: To assess purity (≥85% is considered acceptable for many research applications)
Western blotting: For identity confirmation using specific antibodies
Mass spectrometry: For accurate molecular weight determination and potential post-translational modifications
Circular dichroism: To verify proper protein folding
Size exclusion chromatography: To detect aggregation
Researchers should be vigilant about potential contamination with other proteins, as highlighted by studies showing that recombinant protein impurities can lead to experimental artifacts and misinterpretation of results . Verification from multiple suppliers or production batches can help ensure reproducibility.
Functional characterization of uncharacterized proteins like yjeT requires a multi-faceted approach:
Comparative genomics analysis: Identify conserved domains and potential homologs in other organisms
Interactome studies: Use pull-down assays, yeast two-hybrid, or proximity labeling to identify interaction partners
Gene knockout or knockdown: Assess phenotypic changes in bacteria lacking the protein
Localization studies: Determine subcellular localization using fluorescent tags or fractionation
Structural analysis: Employ X-ray crystallography, cryo-EM, or NMR spectroscopy to determine 3D structure
For transmembrane proteins like yjeT, identifying transport substrates or signaling functions may require specialized assays including liposome reconstitution or electrophysiology experiments. The approach taken for characterizing YjeQ, which was found to be a GTPase with unusual circular permutation of motifs, provides a useful template .
Transmembrane proteins present unique challenges for expression and purification. For yjeT, consider:
Expression optimization:
Testing multiple detergents for solubilization
Using specialized E. coli strains (C41/C43) designed for membrane protein expression
Employing fusion tags that enhance solubility (MBP, SUMO)
Lowering expression temperature to reduce inclusion body formation
Purification refinements:
Screening detergent panels for optimal extraction and stability
Implementing on-column detergent exchange
Using lipid nanodiscs or amphipols for native-like environment maintenance
Applying gentle elution conditions to preserve structure
Stability assessment:
Thermal shift assays with various buffer conditions
Limited proteolysis to identify stable domains
Dynamic light scattering to monitor aggregation
Cell-free expression systems have proven successful for similar transmembrane proteins and could be particularly valuable for yjeT .
When facing contradictory findings regarding yjeT function or properties, implement a systematic approach to resolve discrepancies:
Categorize contradictions: Classify whether contradictions arise from antonymy (direct opposites), negation, numeric mismatches, or structural differences in assertions
Examine experimental conditions: Create a comprehensive comparison table of:
Expression systems used
Purification methods
Buffer compositions
Assay conditions
Detection methods
Consider protein heterogeneity: Assess whether post-translational modifications, alternative conformations, or contaminating proteins could explain divergent results
Control for batch variability: Replicate key experiments with proteins from multiple sources or production batches
Validate with orthogonal methods: Confirm critical findings using independent technical approaches
When analyzing contradictory results, remember that even expression data from the same organism can show poor correlation (correlation coefficients ranging from -0.039 to 0.52) between different experimental conditions , emphasizing the importance of standardized protocols.
Advanced understanding of yjeT could lead to several innovative research applications:
Antimicrobial development: If yjeT proves essential for bacterial survival or virulence, it could become a novel target for antibiotics, particularly valuable if conserved across multiple bacterial species but absent in humans
Biotechnology applications: Potential use as a component in engineered biological systems, such as biosensors or engineered cellular pathways
Structural biology insights: The unique transmembrane architecture might provide new understanding of membrane protein folding and function
Synthetic biology tools: Incorporation into designer protein scaffolds or synthetic cellular circuits
Evolutionary biology research: Investigation of how uncharacterized proteins evolve and potentially acquire new functions
The approach taken with the YjeQ protein, which was found to catalyze GTP hydrolysis at rates 45,000 times greater than turnover , demonstrates how initially uncharacterized proteins can reveal surprising biochemical capabilities when thoroughly investigated.
When facing low yield issues with recombinant yjeT expression:
Optimize codon usage: Adapt codons to the expression host, as codon bias significantly impacts expression levels, especially for membrane proteins
Adjust induction parameters:
Test different inducer concentrations
Modify induction timing (typically at lower OD for membrane proteins)
Evaluate lower temperatures (16-25°C) for extended periods
Enhance solubility:
Screen different fusion tags (His, GST, MBP, SUMO)
Co-express with molecular chaperones
Test specialized E. coli strains
Refine lysis conditions:
Optimize detergent selection and concentration
Evaluate mechanical vs. chemical lysis methods
Add protease inhibitors and reducing agents
Apply DoE approaches: Systematically analyze interactions between critical variables (temperature, media composition, induction time) rather than changing one factor at a time
Implementing rigorous quality control for recombinant yjeT should include:
To distinguish yjeT function from related proteins:
Comparative sequence and structure analysis:
Multiple sequence alignments to identify unique regions
Structural modeling to highlight distinctive features
Phylogenetic analysis to understand evolutionary relationships
Domain-specific studies:
Create chimeric proteins swapping domains between related proteins
Express isolated domains to test for independent functions
Perform site-directed mutagenesis of conserved vs. unique residues
Differential expression analysis:
Compare expression patterns under various conditions
Analyze co-expression networks
Examine regulation mechanisms
Cross-complementation experiments:
Test whether yjeT can complement knockout phenotypes of related proteins
Perform heterologous expression studies across species
Create conditional depletion systems for functional analysis
Interaction network mapping:
Compare protein-protein interaction profiles
Identify unique vs. shared binding partners
Analyze binding affinities and kinetics