Recombinant Yersinia enterocolitica serotype O:8 / biotype 1B UPF0208 membrane protein YE1335 (YE1335)

What is YE1335 protein and what organism does it come from?

YE1335 refers to a UPF0208 membrane protein found in Yersinia enterocolitica serotype O:8/biotype 1B. It is classified as a membrane protein with 151 amino acids in its full-length form . Yersinia enterocolitica is a facultative anaerobic gram-negative rod belonging to the Enterobacteriaceae family, closely related to Yersinia pestis (the causative agent of bubonic plague) . The bacterium is motile with flagella and can survive at refrigerator temperatures, making it a significant foodborne pathogen . In terms of classification, YE1335 has been annotated as a hypothetical protein or a DUF412 domain-containing protein, suggesting its function may not be fully characterized .

How is Yersinia enterocolitica relevant to human health and research?

Yersinia enterocolitica has significant relevance in both clinical and research contexts. This pathogen causes yersiniosis, a gastrointestinal illness that can mimic appendicitis due to its ability to cause mesenteric lymphadenitis and mucosal ulceration of the terminal ileum . The bacterium primarily spreads through contaminated food (especially pork products, poultry, and seafood) and water sources . What makes this organism particularly interesting for research is its pathogenic mechanism – it binds to and invades intestinal epithelial cells, potentially spreading to regional lymph nodes and occasionally causing bacteremia and sepsis . Additionally, it produces an enterotoxin similar to E. coli heat-stable enterotoxin (ST), which contributes to diarrhea symptoms . The organism's ability to grow at refrigeration temperatures and its prolonged shedding in stool (up to 3-4 months after symptom resolution) make it an important subject for food safety and public health research .

What is known about the structure and function of YE1335?

While the search results provide limited specific information on YE1335's function, we can analyze its structural characteristics from the available data. YE1335 is a 151-amino acid membrane protein with the following sequence:
MTTKPSDSVSWFQVLQRGQHYMKTWPADKRLAPVFPENRVATATRFGIRFMPPLAIFTLTWQIALGGQLGPAIATALFACGLPLQGLWWLGKRAITPLPPTLLQWFHEVRNKLAEAGQAVAPVEQTPTYQSLADVLKRAFKQLDKTFLDDL

As a UPF0208 family protein, it belongs to a class of proteins with uncharacterized function. The "DUF412 domain-containing protein" annotation suggests it contains a Domain of Unknown Function 412 . Its classification as a membrane protein indicates it is embedded within or associated with cellular membranes, potentially playing a role in membrane integrity, transport, signaling, or other membrane-associated processes. Research on similar bacterial membrane proteins might focus on their roles in virulence, antibiotic resistance, or bacterial physiology.

What are the available forms of recombinant YE1335 for research?

Several recombinant forms of YE1335 are available for research purposes, differing in expression systems, tags, and coverage of the amino acid sequence:

This diversity of expression systems allows researchers to select the most appropriate form based on their experimental requirements, such as the need for specific post-translational modifications or protein folding conditions.

What expression systems are most effective for producing recombinant YE1335?

When choosing an expression system, researchers should consider:

• E. coli expression: Advantages include high yield, rapid growth, and cost-effectiveness. This system is particularly suitable for proteins that don't require complex post-translational modifications. The successful expression of full-length YE1335 in E. coli suggests that this membrane protein can be properly produced in this system .

• Yeast expression: Provides some eukaryotic post-translational modifications and may offer better folding for certain membrane proteins. This could be beneficial if the bacterial E. coli system produces insoluble or misfolded YE1335.

• Baculovirus/insect cell expression: Offers more complex eukaryotic modifications and can be excellent for membrane proteins that are difficult to express in other systems.

• Mammalian cell expression: Provides the most complex eukaryotic modifications but at higher cost and lower yield. Consider this system if studying YE1335 interactions with mammalian proteins.

• Cell-free expression: Avoids potential toxicity issues and allows for rapid production, which might be advantageous when working with a bacterial membrane protein like YE1335 .

The optimal choice depends on your specific research goals, required protein modifications, downstream applications, and resources.

What purification methods are recommended for recombinant YE1335?

While the search results don't provide detailed purification protocols specific to YE1335, we can infer appropriate methods based on the available recombinant forms. For the His-tagged version of YE1335 , immobilized metal affinity chromatography (IMAC) would be the primary purification method:

Recommended Purification Protocol for His-tagged YE1335:

• Cell Lysis: For E. coli-expressed YE1335, use either sonication, French press, or detergent-based lysis. Since YE1335 is a membrane protein, include appropriate detergents (e.g., n-dodecyl β-D-maltoside (DDM), CHAPS, or Triton X-100) to solubilize the protein from membranes.

• IMAC Purification:

  • Equilibrate Ni-NTA or other metal affinity resin with binding buffer containing detergent

  • Apply clarified lysate to the column

  • Wash with increasing imidazole concentrations to remove non-specific binding

  • Elute His-tagged YE1335 with high imidazole concentration (typically 250-300 mM)

• Secondary Purification:

  • Consider size exclusion chromatography (SEC) to remove aggregates and further purify the protein

  • Ion exchange chromatography may provide additional purification if needed

• Quality Control:

  • Verify purity by SDS-PAGE (should be >90% as specified for commercial preparations)

  • Western blot using anti-His antibodies to confirm identity

  • Mass spectrometry for molecular weight confirmation

For non-His-tagged versions or alternative expression systems, different purification strategies would need to be employed based on the specific tags or properties of the recombinant protein.

How should recombinant YE1335 be stored to maintain stability and activity?

Based on the information provided for commercially available recombinant YE1335, the following storage and handling recommendations should be followed to maintain protein stability and activity:

• Storage Temperature:

  • Store at -20°C or -80°C upon receipt

  • Working aliquots can be stored at 4°C for up to one week

• Physical Form:

  • The protein is typically supplied as a lyophilized powder

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Buffer Conditions:

  • Storage buffer typically consists of Tris/PBS-based buffer with 6% Trehalose, pH 8.0

  • For long-term storage, add glycerol to a final concentration of 5-50% (with 50% being recommended)

• Handling Precautions:

  • Avoid repeated freeze-thaw cycles as they can compromise protein integrity

  • Briefly centrifuge vials before opening to bring contents to the bottom

  • Aliquot reconstituted protein to minimize freeze-thaw cycles

• Working with the Protein:

  • Thaw aliquots on ice or at 4°C

  • As a membrane protein, YE1335 may require detergents to maintain solubility in aqueous solutions

Following these guidelines will help ensure that the recombinant YE1335 maintains its structural integrity and functional properties for your experiments.

How can recombinant YE1335 be used in functional studies?

While the specific function of YE1335 remains uncharacterized, researchers can employ several approaches to investigate its potential roles:

• Membrane Localization Studies:

  • Use fluorescently-tagged YE1335 to visualize its cellular localization

  • Employ subcellular fractionation followed by Western blotting to confirm membrane association

  • Investigate potential lipid raft associations using detergent-resistant membrane isolation

• Protein-Protein Interaction Studies:

  • Perform pull-down assays using His-tagged YE1335 to identify binding partners

  • Use yeast two-hybrid or bacterial two-hybrid systems to screen for interactions

  • Apply proximity labeling techniques (BioID, APEX) to identify proteins in close proximity to YE1335 in its native environment

• Structural Analysis:

  • Conduct circular dichroism (CD) spectroscopy to assess secondary structure elements

  • For high-resolution structure, consider X-ray crystallography or cryo-electron microscopy, though membrane proteins present challenges for these techniques

  • NMR studies on isotopically labeled YE1335 could provide structural insights

• Functional Assays:

  • Generate YE1335 knockout or knockdown in Yersinia enterocolitica to assess phenotypic changes

  • Complement with recombinant YE1335 to confirm specificity of observed effects

  • Investigate potential roles in membrane integrity, transport, or virulence

• Transcriptional Analysis:

  • Examine expression patterns of YE1335 under different growth conditions

  • Identify potential co-regulated genes to infer function

When designing these experiments, researchers should consider the membrane protein nature of YE1335, which may require specialized approaches compared to soluble proteins.

What are the key considerations when designing experiments with YE1335 in relation to Yersinia virulence?

When investigating YE1335's potential role in Yersinia enterocolitica virulence, researchers should consider:

• Pathogenesis Mechanisms: Yersinia enterocolitica causes disease through two primary mechanisms - systemic invasion (binding to and invading intestinal epithelial cells) and enterotoxin production . Design experiments to determine if YE1335 contributes to either pathway.

• Growth Conditions: Y. enterocolitica can grow at refrigeration temperatures , so assess YE1335 expression at different temperatures to understand its potential role in cold adaptation, which contributes to foodborne transmission.

• Cell Infection Models:

  • Use intestinal epithelial cell lines to study potential roles in adhesion and invasion

  • Develop assays measuring bacterial translocation across epithelial monolayers

  • Consider macrophage infection models to assess roles in intracellular survival

• Animal Models:

  • Mouse models can recapitulate aspects of Y. enterocolitica infection

  • Compare wild-type to YE1335 mutant strains for colonization, dissemination to lymph nodes, and disease severity

• Relevant Conditions to Test:

  • pH variation (to mimic gastric passage)

  • Bile salt exposure (intestinal conditions)

  • Iron limitation (host environment)

  • Temperature shifts (environmental to host temperature)

• Potential Virulence Connections:

  • Assess contribution to membrane integrity under stress conditions

  • Investigate roles in antibiotic resistance or efflux

  • Examine potential interactions with known virulence factors

How can contradictory data in YE1335 research be approached methodologically?

When encountering contradictory data in research involving YE1335 or any other protein, a systematic approach is necessary to resolve the inconsistencies:

• Thorough Data Examination:

  • Begin by thoroughly examining your findings to identify specific discrepancies

  • Pay special attention to outliers that might influence results

  • Compare your data with existing literature on similar membrane proteins

• Evaluate Experimental Design:

  • Reassess your initial assumptions and research design

  • Consider whether the contradiction might stem from differences in:

    • Protein preparation methods

    • Buffer conditions

    • Expression systems used (E. coli vs. mammalian)

    • Presence of different tags (His-tag location and size)

    • Full-length vs. partial protein constructs

• Consider Alternative Explanations:

  • Different folding or post-translational modifications across expression systems

  • Potential contamination with bacterial proteins

  • Detergent effects on membrane protein structure/function

  • Batch-to-batch variability in protein preparation

• Refine Variables and Controls:

  • Implement additional controls to isolate specific variables

  • For membrane proteins like YE1335, consider controls such as:

    • Other membrane proteins with similar properties

    • Different detergent conditions

    • Various buffer compositions

    • Protein concentration effects

• Modification of Protocols:

  • Adjust purification methods if protein quality is in question

  • Consider alternative storage conditions (different from the recommended -20°C/-80°C with 50% glycerol)

  • Test different reconstitution protocols beyond the standard recommendation

• Collaborative Verification:

  • Seek independent verification from collaborators using different techniques

  • Consider cross-laboratory validation of key findings

Remember that contradictory data often leads to new insights and can advance understanding of complex biological systems, especially for proteins like YE1335 whose functions remain largely uncharacterized.

What analytical methods are most appropriate for studying membrane protein interactions involving YE1335?

Studying interactions involving membrane proteins like YE1335 requires specialized analytical approaches:

• Co-immunoprecipitation (Co-IP) with Membrane-Specific Adaptations:

  • Use His-tag affinity pulldown with the recombinant His-tagged YE1335

  • Incorporate appropriate detergents to maintain membrane protein solubility

  • Consider crosslinking before lysis to capture transient interactions

  • Control for non-specific binding to detergent micelles

• Surface Plasmon Resonance (SPR):

  • Immobilize purified YE1335 on sensor chips with lipid nanodiscs

  • Measure real-time binding kinetics with potential partner proteins

  • Determine association/dissociation constants (ka, kd, KD)

• Microscale Thermophoresis (MST):

  • Label YE1335 with fluorescent dyes

  • Measure thermophoretic movement in response to potential binding partners

  • Particularly useful for membrane proteins as it works in solution with detergents

• Förster Resonance Energy Transfer (FRET):

  • Create fluorescently labeled YE1335 constructs

  • Measure energy transfer to acceptor-labeled potential interaction partners

  • Can be performed in cellular contexts to verify physiologically relevant interactions

• Biolayer Interferometry:

  • Immobilize His-tagged YE1335 on Ni-NTA biosensors

  • Measure binding in real-time with minimal sample consumption

  • Works well with detergent-solubilized membrane proteins

• Native Mass Spectrometry:

  • Analyze intact protein complexes including membrane proteins in native-like states

  • Requires specialized detergents or nanodiscs for membrane protein analysis

• Isothermal Titration Calorimetry (ITC) Adaptations:

  • Measures thermodynamic parameters of binding

  • Requires careful background subtraction due to detergent effects

When analyzing data from these methods, account for the unique challenges of membrane proteins, including detergent effects, proper control experiments, and potential oligomerization states.

How should unexpected results with YE1335 recombinant protein be troubleshooted?

When working with recombinant YE1335 and encountering unexpected results, a systematic troubleshooting approach is essential:

• Protein Quality Assessment:

  • Verify protein integrity by SDS-PAGE (should show >90% purity for research-grade material)

  • Check for degradation using Western blot with anti-His antibodies

  • Assess aggregation state using size exclusion chromatography or dynamic light scattering

  • Verify protein folding using circular dichroism or limited proteolysis

• Storage and Handling Issues:

  • Ensure proper storage conditions were maintained (-20°C/-80°C)

  • Verify the protein wasn't subjected to repeated freeze-thaw cycles

  • Check if reconstitution was performed according to recommendations (0.1-1.0 mg/mL in deionized sterile water)

  • Confirm appropriate glycerol concentration for long-term storage (recommended 50%)

• Buffer and Reaction Conditions:

  • Test alternative buffer systems beyond the supplied Tris/PBS-based buffer

  • Optimize pH conditions (starting with the recommended pH 8.0)

  • Evaluate different detergents for membrane protein solubility

  • Consider the impact of cofactors or metal ions on activity

• Expression System Considerations:

  • If results differ from expectations, consider testing YE1335 expressed in alternative systems (E. coli vs. eukaryotic systems)

  • Compare results between full-length and partial protein constructs

  • Assess the impact of different tags on protein behavior

• Assay-Specific Troubleshooting:

  • Include positive and negative controls specific to your assay

  • Vary protein concentration to identify optimal working range

  • Consider time-course experiments to capture transient effects

  • Test for interfering substances in your experimental system

• Documentation and Controls:

  • Maintain detailed records of protein lot numbers and preparation methods

  • Use multiple approaches to validate unexpected findings

  • Consider blinding experiments to eliminate unconscious bias

When facing contradictory data, remember that unexpected results often lead to new discoveries, particularly with proteins of unknown function like YE1335 .

What statistical approaches are appropriate for analyzing data from experiments with YE1335?

The appropriate statistical methods for analyzing YE1335 experimental data depend on the type of experiment and data collected. Here are recommended approaches for different experimental scenarios:

• Comparative Studies (e.g., wildtype vs. YE1335 mutant):

  • For normally distributed data: t-tests (paired or unpaired) or ANOVA (for multiple conditions)

  • For non-normally distributed data: Mann-Whitney U test or Kruskal-Wallis test

  • Effect size calculations (Cohen's d) to determine biological significance beyond statistical significance

• Dose-Response Experiments:

  • Nonlinear regression to fit appropriate models (e.g., sigmoidal dose-response)

  • Calculation of EC50/IC50 values with confidence intervals

  • Comparison of curve parameters across experimental conditions

• Time-Course Experiments:

  • Repeated measures ANOVA or mixed-effects models

  • Area under the curve (AUC) analysis

  • Curve fitting to appropriate kinetic models

• Binding and Interaction Studies:

  • Regression analysis to determine binding constants (KD, Bmax)

  • Scatchard or Hill plot analysis for complex binding behaviors

  • Statistical comparison of binding parameters across conditions

• Microscopy and Localization Studies:

  • Quantitative image analysis with appropriate controls

  • Colocalization statistics (Pearson's correlation coefficient, Manders' overlap coefficient)

  • Statistical analysis of distribution patterns

• General Best Practices:

  • Determine appropriate sample sizes through power analysis

  • Apply corrections for multiple comparisons (e.g., Bonferroni, Holm-Sidak, or FDR)

  • Report effect sizes alongside p-values

  • Consider Bayesian approaches for complex datasets

  • Use bootstrapping or permutation tests for non-standard distributions

• Handling Contradictory Data:

  • Meta-analytic approaches to synthesize conflicting results

  • Subgroup analysis to identify factors contributing to heterogeneity

  • Sensitivity analysis to assess robustness of findings

When analyzing data involving membrane proteins like YE1335, pay particular attention to the potential for aggregation, detergent effects, and expression system differences that might introduce variability into your results.

How might YE1335 be involved in Yersinia enterocolitica pathogenesis mechanisms?

While the specific role of YE1335 in Y. enterocolitica pathogenesis is not explicitly described in the search results, we can formulate evidence-based hypotheses based on its membrane localization and the known pathogenesis mechanisms of the organism:

• Potential Involvement in Cellular Invasion:

  • Y. enterocolitica binds to and invades intestinal epithelial cells

  • As a membrane protein, YE1335 could potentially mediate interactions with host cell receptors

  • It might form part of a membrane complex involved in adhesion or invasion processes

  • Research approaches could include comparing invasion efficiency between wildtype and YE1335 mutant strains

• Possible Role in Environmental Adaptation:

  • Y. enterocolitica can grow at refrigerator temperatures , suggesting specialized membrane adaptations

  • YE1335 might contribute to membrane fluidity regulation under different temperature conditions

  • Expression analysis of YE1335 at different temperatures could provide insights into this potential role

• Hypothetical Functions in Resistance Mechanisms:

  • Membrane proteins often participate in efflux systems or permeability barriers

  • YE1335 might contribute to survival in hostile host environments (e.g., acidic pH, antimicrobial peptides)

  • Susceptibility testing of YE1335 mutants to various stressors could reveal protective functions

• Potential Contribution to Virulence Regulation:

  • Membrane proteins can function as sensors for environmental cues

  • YE1335 might participate in signaling pathways that regulate virulence gene expression

  • Transcriptional profiling of YE1335 mutants under infection-relevant conditions could identify regulatory roles

• Possible Involvement in Enterotoxin Secretion:

  • Y. enterocolitica produces an enterotoxin similar to E. coli heat-stable enterotoxin

  • YE1335 could potentially be involved in toxin transport or secretion

  • Toxin quantification in YE1335 mutant culture supernatants could test this hypothesis

These hypothetical functions would need to be tested experimentally, starting with the creation of YE1335 knockout strains and phenotypic characterization under various conditions relevant to pathogenesis.

What cutting-edge approaches could be applied to determine the function of the uncharacterized YE1335 protein?

Revealing the function of uncharacterized proteins like YE1335 requires innovative techniques that go beyond traditional approaches:

• Cryo-Electron Microscopy (Cryo-EM):

  • Apply single-particle cryo-EM to determine the high-resolution structure of YE1335

  • Consider lipid nanodisc reconstitution to maintain a native-like membrane environment

  • Structural insights can provide functional clues through identification of potential binding sites or functional domains

• AlphaFold2 and Computational Structure Prediction:

  • Use AI-based structure prediction to generate models of YE1335

  • Compare predicted structure to known membrane proteins to identify functional analogues

  • Molecular dynamics simulations can reveal dynamic properties and potential functional sites

• CRISPR Interference (CRISPRi) and CRISPR Activation (CRISPRa):

  • Apply for fine-tuned modulation of YE1335 expression rather than complete knockout

  • Identify phenotypic effects of varying expression levels under different conditions

  • Combine with transcriptomic analysis to identify compensatory responses

• Thermal Proteome Profiling (TPP):

  • Identify potential ligands or interaction partners through shifts in YE1335 thermal stability

  • Apply to whole cells to identify targets in their native environment

  • Can reveal unexpected functions through identification of stabilizing molecules

• Proximity-Dependent Biotin Identification (BioID or TurboID):

  • Fuse biotin ligase to YE1335 to biotinylate proximal proteins in vivo

  • Identify the neighborhood of proteins surrounding YE1335 in the membrane

  • Provides context for potential functional roles in larger complexes

• Lipidomics Analysis with YE1335 Variants:

  • Compare lipid profiles between wildtype and YE1335 mutant strains

  • Investigate potential roles in membrane lipid organization or metabolism

  • Apply to different growth conditions relevant to the Y. enterocolitica lifecycle

• Single-Cell Techniques:

  • Apply single-cell RNA-seq to identify transcriptional heterogeneity in response to YE1335 modulation

  • Use single-cell microscopy with fluorescent reporters to track dynamic processes

  • Reveal population heterogeneity that might be masked in bulk assays

These advanced approaches can be combined in an integrative strategy to build converging evidence for YE1335 function, leading to a more comprehensive understanding of this uncharacterized protein's role in Yersinia enterocolitica.

How might recombinant YE1335 be utilized in developing new antimicrobial strategies against Yersinia infections?

The recombinant YE1335 protein could be leveraged in several innovative approaches to developing antimicrobial strategies:

• Structural Vaccinology Approach:

  • Use purified recombinant YE1335 to identify immunogenic epitopes

  • Design peptide vaccines based on exposed regions of the protein

  • If YE1335 proves to be surface-exposed and conserved across pathogenic Yersinia strains, it could serve as a vaccine antigen

• Drug Target Validation:

  • If functional studies reveal YE1335 to be essential for virulence or survival

  • Establish high-throughput screening assays using recombinant YE1335

  • Screen for small molecule inhibitors that bind specifically to YE1335

• Antibody-Based Therapeutics:

  • Generate monoclonal antibodies against recombinant YE1335

  • Evaluate their ability to neutralize bacterial virulence

  • Develop antibody-antibiotic conjugates for targeted delivery

• Phage Display for Peptide Inhibitors:

  • Screen phage display libraries against recombinant YE1335

  • Identify peptides that bind with high affinity and specificity

  • Develop peptide-based inhibitors of YE1335 function

• Structure-Based Drug Design:

  • Use the amino acid sequence and structural predictions to model YE1335

  • Identify potential binding pockets through computational analysis

  • Design small molecules that could interfere with essential functions

• Adjuvant Development:

  • Investigate whether YE1335 has immunomodulatory properties

  • If so, explore its potential as an adjuvant for other vaccines

  • Test various formulations of recombinant YE1335 for adjuvant activity

• Diagnostic Development:

  • Use recombinant YE1335 to generate specific antibodies

  • Develop rapid diagnostic tests for Yersinia enterocolitica detection

  • Create multiplex assays distinguishing pathogenic serotypes like O:8

These approaches would require thorough validation of YE1335's role in pathogenesis, as the search results don't provide direct evidence for its function in virulence. The development process would need to consider differences between the recombinant protein expressed in E. coli versus its native form in Y. enterocolitica.