Recombinant Uncharacterized protein yjeT (yjeT)

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Description

Introduction to Recombinant Uncharacterized Protein YjeT

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.

Production and Physical Properties of Recombinant YjeT

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

ParameterSpecification
OrganismEscherichia coli
Expression SystemEscherichia coli
Fusion TagN-terminal His tag
Protein LengthFull Length (amino acids 1-65)
Physical FormLyophilized powder
Purity LevelGreater than 90% (SDS-PAGE verified)
UniProt IdentifierP0AF74
Gene SynonymsYjeT; Z5783; ECs5152
Amino Acid SequenceMNSTIWLALALVLVLEGLGPMLYPKAWKKMISAMTNLPDNILRRFGGGLVVAGVVVYYMLRKTIG

This information, derived from product specifications provided by Creative Biomart, outlines the key characteristics of commercially available recombinant YjeT protein .

Reconstitution Protocol for Lyophilized YjeT

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

StepProcedure
1Centrifuge the vial briefly prior to opening
2Reconstitute in deionized sterile water to 0.1-1.0 mg/mL
3For extended storage, add glycerol to 5-50% final concentration
4Prepare aliquots to minimize freeze-thaw cycles
5Store 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.

Current Research Status on YjeT Function

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.

Research Applications of Recombinant YjeT

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 ApproachPotential Application
Structural AnalysisDetermination of three-dimensional structure through X-ray crystallography or NMR spectroscopy
Functional AssaysBiochemical tests to identify potential enzymatic activities or cellular functions
Protein-Protein Interaction StudiesIdentification of binding partners to infer functional roles
Localization StudiesDetermination of subcellular localization to suggest functional context
Comparative AnalysisComparison 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.

Context from Related Bacterial Protein Research

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.

Future Research Directions for YjeT Characterization

Based on the limited current understanding of YjeT, several research directions would be valuable for advancing knowledge about this protein:

  1. Comprehensive functional characterization through genetic approaches, including gene deletion and complementation studies to determine essentiality and phenotypic effects

  2. Structural determination through advanced techniques to identify potential functional domains and binding sites

  3. Proteomic analyses to identify potential interaction partners and protein complexes involving YjeT

  4. Comparative genomic studies to understand evolutionary conservation and potential functional importance across bacterial species

  5. 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.

Product Specs

Form
Lyophilized powder
Note: While we prioritize shipping the format currently in stock, please specify your format preference during order placement for customized preparation.
Lead Time
Delivery times vary depending on the purchasing method and location. Please contact your local distributor for precise delivery estimates.
Note: All proteins are shipped with standard blue ice packs. Dry ice shipping requires prior arrangement and incurs additional charges.
Notes
Avoid repeated freeze-thaw cycles. Store working aliquots at 4°C for up to one week.
Reconstitution
Centrifuge the vial briefly before opening to consolidate the contents. Reconstitute the protein in sterile, deionized water to a concentration of 0.1-1.0 mg/mL. We recommend adding 5-50% glycerol (final concentration) and aliquoting for long-term storage at -20°C/-80°C. Our standard glycerol concentration is 50% and serves as a guideline.
Shelf Life
Shelf life depends on several factors, including storage conditions, buffer composition, temperature, and protein stability. Generally, liquid formulations have a 6-month shelf life at -20°C/-80°C, while lyophilized forms have a 12-month shelf life at -20°C/-80°C.
Storage Condition
Upon receipt, store at -20°C/-80°C. Aliquoting is essential for multiple uses. Avoid repeated freeze-thaw cycles.
Tag Info
Tag type is determined during the manufacturing process.
The tag type is determined during production. If you require a specific tag, please inform us, and we will prioritize its development.
Synonyms
yjeT; SF4331; S4599; Uncharacterized protein YjeT
Buffer Before Lyophilization
Tris/PBS-based buffer, 6% Trehalose.
Datasheet
Please contact us to get it.
Expression Region
1-65
Protein Length
full length protein
Species
Shigella flexneri
Target Names
yjeT
Target Protein Sequence
MNSTIWLALALVLVLEGLGPMLYPKAWKKMISAMTNLPDNILRRFGGGLVVAGVVVYYML RKTIG
Uniprot No.

Target Background

Database Links

KEGG: sfl:SF4331

Subcellular Location
Cell membrane; Multi-pass membrane protein.

Q&A

What is yjeT protein and why is it significant for research?

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 .

What expression systems are most suitable for producing recombinant yjeT protein?

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.

How can Design of Experiments (DoE) approach improve yjeT protein purification?

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 .

FactorLow LevelHigh LevelPrimary Effect on Yield
pH6.08.0Moderate
Temperature4°C30°CHigh
Salt concentration0.1M0.5MModerate
Additive concentration0%0.5%High

What purification strategy would be most effective for recombinant yjeT?

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 .

How can researchers verify the purity and integrity of recombinant yjeT protein?

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.

How can researchers approach functional characterization of uncharacterized proteins like yjeT?

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 .

What strategies can address the challenges of expressing and purifying transmembrane proteins like yjeT?

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 .

How can contradictions in experimental data about yjeT be systematically analyzed and resolved?

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.

What novel applications might be developed through better understanding of yjeT protein?

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.

How can researchers overcome low yield issues when expressing recombinant yjeT?

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

What quality control measures are essential when working with recombinant yjeT protein?

Implementing rigorous quality control for recombinant yjeT should include:

How should researchers design experiments to differentiate between the functions of yjeT and related uncharacterized proteins?

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

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