YjeT is typically expressed in E. coli using plasmid vectors with affinity tags for purification. Common systems include:
Low solubility requiring optimization (e.g., glycerol additives, buffer pH 8.0) .
Stability issues necessitating storage at -80°C in Tris/PBS buffers with 6% trehalose .
Property | Value |
---|---|
Purity | >90% (SDS-PAGE) |
Storage Buffer | Tris/PBS, 50% glycerol, pH 8.0 |
Storage Temperature | -80°C (long-term); 4°C (working aliquots) |
No enzymatic activity (e.g., GTPase/ATPase) has been detected, unlike YjeQ .
No confirmed interactions with ribosomal subunits or nucleic acids, despite homology to RNA-binding OB-fold proteins .
YjeT is primarily used as a reagent in:
KEGG: ecj:JW4134
STRING: 316385.ECDH10B_4371
YjeT is classified as an uncharacterized conserved protein belonging to the DUF2065 (Domain of Unknown Function) family under COG3242 in the "Function unknown" (S) category . As an uncharacterized protein, YjeT represents one of many hypothetical proteins (HPs) that are predicted to be expressed from an open reading frame but have not been experimentally characterized for structure or function . The protein is part of the E. coli proteome, and like many uncharacterized proteins, it requires systematic investigation to determine its biological role.
For the expression of uncharacterized proteins like YjeT, E. coli remains the host of choice due to its fast growth, easy manipulation, and cost-effectiveness . The BL21(DE3) strain is particularly preferred for recombinant protein production because it has several advantageous features:
For proteins that require disulfide bond formation, consider targeting to the periplasm using appropriate signal peptides to take advantage of the oxidizing environment and Dsb-system .
Verification of YjeT expression should employ multiple complementary techniques:
SDS-PAGE Analysis: Separates proteins according to molecular weight, allowing comparison with marker proteins to confirm the expected size of YjeT .
Western Blotting: If antibodies are available or if YjeT is expressed with a tag (e.g., His-tag), this technique provides specific detection.
Mass Spectrometry: For unambiguous identification, peptide mass fingerprinting or Tandem MS (MS-MS) approaches can confirm the identity of YjeT by matching experimentally obtained masses to theoretical peptide masses .
Database Verification: Tools like YPED (Yale Protein Expression Database) can help with comparing MS data to validate the expression of YjeT against existing spectral libraries .
Optimizing YjeT expression requires a systematic approach addressing several parameters:
Signal Peptide Selection: For periplasmic targeting, perform a combinatorial screen of different signal peptides, as this can significantly enhance protein yields .
Expression Rate Control: Adjusting the production rate by modulating promoter strength or inducer concentration can prevent aggregation of overexpressed proteins .
Host Strain Selection: Consider specialized strains like BL21(DE3) derivatives that address specific expression challenges:
Culture Conditions: Maintain pH between 7.5-8.5 to minimize acetate stress, which improves recombinant protein production .
The purification strategy should be tailored to YjeT's properties and downstream applications:
For efficient purification, a multi-step approach is recommended, beginning with an affinity-based method (if a tag is used), followed by ion-exchange for removing impurities, and concluding with gel filtration for final purity assessment and buffer exchange.
Characterizing the structure of YjeT requires multiple complementary approaches:
X-ray Crystallography: Provides high-resolution structural information if crystals of purified YjeT can be obtained.
NMR Spectroscopy: Useful for analyzing YjeT's structure in solution and potential dynamic properties.
Cryo-EM: Emerging technique for structural analysis, particularly valuable if YjeT forms larger complexes.
Bioinformatic Structure Prediction: Tools like AlphaFold can predict structures of uncharacterized proteins, which can guide experimental approaches.
Circular Dichroism (CD) Spectroscopy: Provides information about YjeT's secondary structure content (α-helices, β-sheets).
Functional characterization requires systematic investigation using multiple approaches:
Bioinformatic Analysis:
Sequence similarity searches to identify homologs with known functions
Domain identification to infer potential biochemical activities
Genomic context analysis to identify operons or functionally related genes
Protein-Protein Interaction Studies:
Gene Knockout Studies:
Create yjeT deletion mutants and assess phenotypic changes
Complement the knockout with wild-type or mutated versions
Expression Analysis:
Determine conditions under which yjeT is expressed using RNA-seq or qPCR
Analyze regulation using promoter-reporter fusions
Enzymatic Activity Assays:
Screen for potential enzymatic activities based on structural predictions
Test for activities common to the protein family or genomic neighbors
If YjeT forms inclusion bodies, consider these strategies:
Optimization of Expression Conditions:
Lower temperature (16-25°C)
Reduce inducer concentration
Use weaker promoters
Co-express with molecular chaperones
Fusion Tags to Enhance Solubility:
MBP (Maltose Binding Protein)
SUMO
Thioredoxin
NusA
Inclusion Body Processing:
Develop a refolding protocol specific to YjeT
Use high-throughput screening of refolding conditions
Consider on-column refolding during purification
Periplasmic Expression:
Signal Peptide | Production Rate | Periplasmic Yield | Inclusion Body Formation |
---|---|---|---|
DsbA | High | Low | High |
DsbA | Low | Moderate | Low |
PelB | High | Moderate | Moderate |
PelB | Low | High | Very Low |
OmpA | Low | Moderate | Low |
To investigate YjeT interactions:
Crosslinking Mass Spectrometry:
Use chemical crosslinkers to capture transient interactions
Identify interaction partners through LC-MS/MS analysis
Co-evolution Analysis:
Computational methods to predict functional associations based on evolutionary patterns
Bacterial Two-Hybrid Systems:
Modified for use in prokaryotic systems to detect protein-protein interactions
Fluorescence Microscopy:
Tag YjeT with fluorescent proteins to observe localization
Use FRET to detect interactions with tagged potential partners
Microfluidics Approaches:
Validation requires multiple approaches:
Biophysical Characterization:
Circular dichroism to assess secondary structure
Thermal shift assays to evaluate stability
Dynamic light scattering to assess homogeneity
Activity Assays:
Design based on bioinformatic predictions
Test for common activities in the protein family
Develop reporter systems for potential functions
Structural Integrity:
Limited proteolysis to assess compact folding
NMR 1D spectra to evaluate tertiary structure
In vivo Complementation:
Test if the purified protein can restore function in knockout strains
Robust statistical analysis is crucial for YjeT research:
Beyond Simple Significance Testing:
For Structural Studies:
Apply appropriate model validation statistics
Report resolution and refinement statistics for crystallography
Use multiple scoring functions for computational models
For Functional Assays:
Use appropriate statistical tests based on data distribution
Report variability (standard deviation, standard error)
Consider biological replicates vs. technical replicates
For Omics Data Integration:
Designing appropriate controls is essential:
Expression and Purification Controls:
Empty vector control
Well-characterized protein expressed under identical conditions
Tag-only expression control
Functional Assay Controls:
Positive controls with known activity
Negative controls (heat-inactivated protein, catalytic mutants)
Buffer controls to identify buffer component effects
Interaction Study Controls:
Unrelated proteins to test for non-specific binding
Competition assays with unlabeled proteins
Proper negative controls for two-hybrid systems
In vivo Study Controls:
Wild-type strain
Knockout strain
Complemented strain with wild-type gene
Complemented strain with mutated gene
A comprehensive methodology section should include:
Detailed Protocols:
Provide sufficient detail for replication by other researchers3
Include all reagents, conditions, and equipment specifications
Data Collection Methods:
Describe sampling methods and criteria for participant/sample selection3
Detail tools, procedures, and materials used
Analysis Methods:
Describe data preparation and software used3
Specify statistical methods applied
Methodological Justification:
Explain why chosen methods were appropriate3
Acknowledge limitations of the approach
Data Presentation:
Characterizing YjeT has several potential implications:
Fundamental Knowledge Gaps:
Evolutionary Perspectives:
Functional Networks:
Identification of YjeT function may reveal new connections in cellular networks
Could fill gaps in our understanding of bacterial physiology
Emerging technologies offer new opportunities:
Cryo-Electron Tomography:
Visualize proteins in their native cellular context
May reveal YjeT localization and interactions in intact cells
Single-Cell Proteomics:
Analyze YjeT expression at single-cell resolution
Reveal cell-to-cell variability in expression or modification
Synthetic Biology Approaches:
Create reporter systems to detect YjeT activity
Engineer strains with controlled expression for functional studies
Genome-Wide Interaction Screens:
CRISPR interference screens to identify genetic interactions
Transposon sequencing (Tn-seq) to identify synthetic lethal interactions
Integrative Computational Approaches:
Combine multiple omics datasets to predict function
Apply machine learning to identify patterns across diverse data types