Recombinant Enterococcus faecalis UPF0758 protein EF_2926 (EF_2926)

What is Recombinant Enterococcus faecalis UPF0758 protein EF_2926?

Recombinant Enterococcus faecalis UPF0758 protein EF_2926 is a full-length protein (232 amino acids) originating from Enterococcus faecalis strain ATCC 700802/V583. It is typically expressed in E. coli expression systems and has the UniProt accession number Q82ZX1. The protein belongs to the UPF0758 protein family with currently uncharacterized function. The recombinant form available commercially has a purity of >85% as determined by SDS-PAGE analysis .

What is currently known about the function of EF_2926?

While EF_2926 is classified as a UPF0758 family protein, its specific biological function remains largely uncharacterized. Researchers should note that Enterococcus faecalis has been studied for its synergistic virulent effects with E. coli in polymicrobial infections . Investigating whether EF_2926 plays a role in these interactions could be a valuable research direction. Preliminary studies suggest potential involvement in bacterial stress response mechanisms, but further functional characterization through methods such as gene knockout studies, protein-protein interaction analyses, and structural determination would provide greater insight.

What are the optimal storage conditions for maintaining EF_2926 stability?

The stability and shelf life of Recombinant EF_2926 depend on multiple factors including storage state, buffer ingredients, and storage temperature. For liquid preparations, the recommended storage is at -20°C/-80°C with an expected shelf life of approximately 6 months. Lyophilized forms demonstrate extended stability of up to 12 months when stored at -20°C/-80°C. Importantly, repeated freeze-thaw cycles should be avoided as they can significantly compromise protein integrity. For short-term usage (up to one week), working aliquots may be stored at 4°C .

What is the recommended reconstitution procedure for lyophilized EF_2926?

For optimal reconstitution of lyophilized EF_2926:

• Briefly centrifuge the vial prior to opening to ensure all content is at the bottom

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

• Add glycerol to a final concentration of 5-50% (50% is commonly recommended)

• Create multiple small-volume aliquots to minimize freeze-thaw cycles

• Store reconstituted aliquots at -20°C/-80°C for long-term storage

This methodology preserves protein activity and prevents degradation during storage periods.

How should Design of Experiments (DoE) be applied when optimizing expression conditions for EF_2926?

Designing experiments to optimize EF_2926 expression should employ DoE approaches rather than the inefficient one-factor-at-a-time method. The DoE strategy enables researchers to evaluate multiple factors simultaneously, including temperature, induction timing, inducer concentration, and media composition. This approach identifies not only individual factor effects but also their interactions, which is crucial for recombinant protein optimization .

A recommended DoE framework for EF_2926 expression includes:

• Factor identification: Select 3-5 key variables most likely to affect expression (e.g., temperature, IPTG concentration, post-induction time)

• Range determination: Establish appropriate ranges for each factor based on literature and preliminary experiments

• Experimental design selection: Implement either fractional factorial designs (for screening many factors) or response surface methodology (for optimization of a few factors)

• Response measurement: Quantify protein yield and quality (purity, activity) as response variables

• Statistical analysis: Use available software packages to analyze results and identify optimal conditions

• Confirmation runs: Validate the predicted optimal conditions with confirmation experiments

This methodical approach minimizes experimental costs while maximizing information gained about EF_2926 production conditions.

What controls should be included when studying EF_2926 interactions with other proteins?

When investigating EF_2926 interactions with other proteins, particularly in the context of its potential role in Enterococcus faecalis virulence, several controls are essential:

• Negative interaction controls: Include non-relevant proteins of similar size/structure to rule out non-specific binding

• Binding buffer controls: Test multiple buffer conditions to ensure interactions are not artifacts of experimental conditions

• Tag-only controls: If tagged EF_2926 is used, include the tag alone to eliminate tag-mediated interactions

• Concentration gradients: Test multiple protein concentrations to establish dose-dependency of interactions

• Competitive inhibition: Use excess unlabeled protein to verify binding site specificity

• Domain deletion variants: Use truncated versions of EF_2926 to map interaction domains

Given the potential virulent synergistic effect of Enterococcus faecalis with other bacteria like E. coli , interactions between EF_2926 and proteins from potential synergistic partners should be systematically evaluated using these control measures.

What expression systems are most effective for producing high-yield, functional EF_2926?

• Strain selection: BL21(DE3) and its derivatives are commonly preferred for their reduced protease activity and tight expression control

• Codon optimization: Adapting the EF_2926 gene sequence to E. coli codon usage can significantly enhance expression levels

• Expression vectors: pET system vectors containing T7 promoters generally provide high expression levels for prokaryotic proteins

• Solubility enhancement: Fusion partners such as MBP, SUMO, or Thioredoxin may improve solubility if inclusion body formation is observed

• Induction conditions: Lower temperatures (16-25°C) and reduced inducer concentrations often favor proper folding over high-speed production

For researchers requiring post-translational modifications not available in E. coli systems, yeast-based systems (Pichia pastoris) may be considered, though this would require extensive optimization through DoE approaches .

What purification strategy is recommended for obtaining high-purity EF_2926?

A multi-step purification strategy is recommended to achieve >95% purity for sensitive applications:

• Initial capture: Affinity chromatography using an appropriate tag (His-tag is common for recombinant proteins)

• Intermediate purification: Ion exchange chromatography based on EF_2926's theoretical pI

• Polishing step: Size exclusion chromatography to remove aggregates and achieve final purity

• Quality control: Validate purity using SDS-PAGE, Western blot, and mass spectrometry

• Endotoxin removal: If intended for cell culture or in vivo applications, additional endotoxin removal steps

For researchers working with the commercially available recombinant EF_2926, note that the product specifications indicate a purity of >85% by SDS-PAGE . Additional purification steps may be necessary depending on experimental requirements.

What methodologies are most informative for characterizing the biochemical properties of EF_2926?

To comprehensively characterize EF_2926's biochemical properties, employ these methodological approaches:

• Structural analysis:

  • Circular Dichroism (CD) spectroscopy for secondary structure determination

  • X-ray crystallography or NMR for high-resolution structural information

  • Thermal shift assays to assess stability under various conditions

• Functional assessments:

  • ATPase activity assays (if ATP binding motifs are present)

  • DNA/RNA binding assays (electrophoretic mobility shift assays)

  • Phosphorylation state analysis via mass spectrometry

  • Enzymatic activity screens based on sequence homology predictions

• Interaction studies:

  • Pull-down assays to identify binding partners

  • Surface Plasmon Resonance (SPR) for binding kinetics

  • Isothermal Titration Calorimetry (ITC) for thermodynamic parameters

These methods should be applied systematically, starting with structural characterization to inform functional hypotheses.

How can researchers investigate the potential role of EF_2926 in Enterococcus faecalis virulence?

Given the known virulent synergistic effect between Enterococcus faecalis and E. coli in polymicrobial infections , investigating EF_2926's potential role in virulence requires a multi-faceted approach:

• Genetic approaches:

  • Generate knockout mutants lacking EF_2926 and assess virulence in appropriate models

  • Complement mutants with wild-type and mutated versions to confirm specificity

  • Perform transcriptomic analysis comparing wild-type and mutant strains under infection-relevant conditions

• Protein interaction studies:

  • Identify host cell targets using pull-down assays with host cell lysates

  • Verify interactions using co-immunoprecipitation and co-localization studies

  • Test for direct interaction with virulence factors from synergistic bacterial partners

• Infection models:

  • Evaluate Caenorhabditis elegans survival rates as described in previous studies

  • Test polymicrobial infections with wild-type and EF_2926-deficient strains

  • Monitor virulence parameters including:

    • Host survival rates

    • Bacterial burden

    • Inflammatory response markers

    • Tissue damage assessment

The research should compare EF_2926's role in mono-species vs. polymicrobial infections to elucidate its contribution to the synergistic virulence observed between Enterococcus faecalis and other bacteria.

What strategies can resolve poor expression yields of recombinant EF_2926?

Poor expression yields of EF_2926 may result from multiple factors. Apply these systematic troubleshooting approaches:

• Optimize expression conditions using DoE:

  • Test temperature ranges (16°C, 25°C, 30°C, 37°C)

  • Vary inducer concentrations (IPTG: 0.1mM to 1mM range)

  • Adjust post-induction times (3h, 6h, overnight)

  • Evaluate different media formulations (LB, TB, auto-induction media)

• Address potential toxicity issues:

  • Test expression in different E. coli strains (BL21, C41/C43 for toxic proteins)

  • Use tightly controlled promoters (araBAD, rhamnose-inducible)

  • Reduce basal expression by including glucose in pre-induction media

• Improve protein solubility:

  • Co-express with molecular chaperones (GroEL/ES, DnaK/J)

  • Add solubility-enhancing fusion tags (MBP, SUMO, TrxA)

  • Include mild solubilizing agents in lysis buffer (0.1% Triton X-100, 5% glycerol)

• Check for potential degradation:

  • Add protease inhibitors during purification

  • Perform Western blot analysis to identify degradation products

  • Optimize cell lysis conditions to minimize proteolytic activity

A structured Design of Experiments approach will allow researchers to identify optimal conditions more efficiently than changing one factor at a time .

How can researchers address stability issues with purified EF_2926?

Stability issues with purified EF_2926 can significantly impact experimental outcomes. Implement these methodological solutions:

• Buffer optimization:

  • Screen multiple buffer compositions using differential scanning fluorimetry

  • Test pH ranges from 6.0-8.0 in 0.5 increments

  • Evaluate stabilizing additives:

    • Glycerol (5-20%)

    • NaCl or KCl (50-500mM)

    • Reducing agents (DTT, TCEP, β-mercaptoethanol)

    • Divalent cations (Mg²⁺, Ca²⁺) at 1-5mM

• Storage protocol refinement:

  • Prepare small aliquots to avoid freeze-thaw cycles

  • Add glycerol to a final concentration of 50% for -20°C storage

  • Consider lyophilization for long-term storage (extends shelf life to 12 months)

  • For working stocks, limit 4°C storage to one week maximum

• Analytical techniques to monitor stability:

  • Size exclusion chromatography to assess aggregation state

  • Dynamic light scattering to detect early aggregation events

  • Activity assays (once established) to confirm functional integrity

  • SDS-PAGE under non-reducing and reducing conditions to monitor disulfide bond status

Implementing these approaches in a systematic manner will help identify the optimal conditions for maintaining EF_2926 stability throughout experimental workflows.

How might EF_2926 contribute to the synergistic virulence observed in polymicrobial infections?

Research into polymicrobial infections has demonstrated a synergistic virulent effect between Enterococcus faecalis and E. coli, significantly reducing the time to mortality in experimental models (LT50 = 1.6 days for the polymicrobial infection versus 4.6 days for Enterococcus faecalis alone) . While the specific role of EF_2926 in this synergy has not been fully characterized, several hypothetical mechanisms can be investigated:

• Interspecies signaling:

  • EF_2926 may function in quorum sensing or interspecies communication

  • Test whether purified EF_2926 alters E. coli gene expression patterns

  • Evaluate if EF_2926 influences biofilm formation in mixed-species communities

• Host defense modulation:

  • Investigate if EF_2926 suppresses host immune responses

  • Determine whether it synergizes with E. coli virulence factors

  • Assess its impact on host epithelial barrier integrity

• Metabolic cooperation:

  • Examine if EF_2926 facilitates cross-feeding between bacterial species

  • Test for enzymatic activities that modify the infection microenvironment

  • Evaluate its role in adaptation to host-imposed nutritional immunity

These research directions should be explored using both in vitro co-culture systems and in vivo infection models, with appropriate genetic manipulations of the EF_2926 gene to establish causality.

What structural biology approaches would be most informative for understanding EF_2926 function?

Elucidating the structure of EF_2926 is crucial for understanding its function. The following structural biology approaches are recommended:

• X-ray crystallography workflow:

  • High-throughput crystallization condition screening

  • Optimization of crystal growth parameters

  • Data collection at synchrotron radiation facilities

  • Structure determination using molecular replacement or experimental phasing

  • Refinement and validation of the structural model

• NMR spectroscopy studies:

  • Express isotopically labeled protein (¹⁵N, ¹³C)

  • Collect 2D and 3D NMR spectra for backbone assignment

  • Analyze chemical shift perturbations upon ligand binding

  • Identify dynamic regions that may be functionally important

• Cryo-electron microscopy:

  • Particularly valuable if EF_2926 forms larger complexes

  • Sample vitrification optimization

  • Image acquisition and processing

  • 3D reconstruction and model building

• Computational approaches:

  • Homology modeling based on structural homologs

  • Molecular dynamics simulations to predict flexibility

  • Virtual screening for potential binding partners

  • Structure-based functional annotation

The structural data should be integrated with biochemical and genetic studies to develop comprehensive models of EF_2926 function within Enterococcus faecalis biology and potential roles in virulence.