Recombinant Enterococcus faecalis Probable transcriptional regulatory protein EF_2866 (EF_2866)

What is Enterococcus faecalis transcriptional regulatory protein EF_2866?

EF_2866 is classified as a probable transcriptional regulatory protein encoded in the genome of Enterococcus faecalis strain ATCC 700802/V583. The protein consists of 239 amino acids and functions as a regulatory element likely involved in controlling gene expression patterns in E. faecalis. The protein has been assigned the UniProt accession number Q830C3, indicating its cataloging in standard protein databases . As a transcriptional regulator, EF_2866 likely binds to specific DNA sequences to influence the expression of target genes, though the specific regulatory networks and binding targets remain areas of active investigation.

What are the recommended storage conditions for recombinant EF_2866?

The stability and activity of recombinant EF_2866 depend significantly on proper storage conditions. For liquid formulations, the recommended storage is at -20°C/-80°C, which provides a typical shelf life of approximately 6 months. For lyophilized formulations, storage at -20°C/-80°C extends the shelf life to approximately 12 months .

To minimize protein degradation from freeze-thaw cycles, it is advisable to 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% (with 50% being standard practice), and create working aliquots for long-term storage. For short-term use (up to one week), working aliquots can be stored at 4°C . Repeated freezing and thawing should be avoided as this can lead to protein denaturation and loss of activity.

What expression systems are typically used for EF_2866 production?

Recombinant EF_2866 is typically produced using E. coli expression systems . This heterologous expression approach is preferred due to several advantages:

• High yield of target protein

• Well-established protocols for transformation and induction

• Scalability for laboratory research purposes

• Compatibility with various purification tags

When expressing EF_2866 in E. coli, researchers commonly employ vectors that allow for the addition of purification tags, facilitating subsequent protein isolation. The expression region typically encompasses the full-length protein (amino acids 1-239) . Selection of appropriate E. coli strains is crucial for optimal expression, with BL21(DE3) and its derivatives being common choices due to their reduced protease activity.

How can purity of recombinant EF_2866 be assessed?

The purity of recombinant EF_2866 is typically assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with acceptance criteria generally set at >85% purity . This analytical method separates proteins based on molecular weight, allowing visualization of the target protein and potential contaminants.

For a comprehensive purity assessment, the following methodological approach is recommended:

• Prepare protein samples by mixing with Laemmli buffer containing a reducing agent

• Heat samples at 95°C for 5 minutes to ensure complete denaturation

• Load samples alongside molecular weight markers onto 10-15% polyacrylamide gels

• Run electrophoresis at constant voltage (typically 120-150V)

• Stain gels with Coomassie Blue or silver stain

• Quantify band intensity using densitometry software

Additional purity analyses may include:

• Western blotting with anti-EF_2866 antibodies

• Size exclusion chromatography (SEC)

• Mass spectrometry to confirm protein identity and detect modifications

What proteomic approaches can be used to study EF_2866?

Data-independent acquisition mass spectrometry (DIA-MS) represents a powerful proteomic approach for studying EF_2866 and its interactions within the Enterococcus faecalis proteome. When implementing DIA-MS for EF_2866 analysis, the following methodological parameters should be considered:

• Sample preparation: Set False Discovery Rate (FDR) at 1% on spectral, peptide, and protein group levels

• Quantification parameters: Utilize up to 6 fragments per peptide and up to 10 peptides per protein

• Data processing: Implement dynamic retention time alignment, dynamic mass recalibration, and quartile normalization for 1% FDR

• Data analysis: Perform global data imputation for the final results table

This approach can reveal EF_2866's expression patterns under different conditions, potential post-translational modifications, and protein-protein interactions. When comparing EF_2866 expression across different experimental conditions, researchers should apply appropriate statistical analyses to identify significant changes in protein abundance.

How does EF_2866 compare to other transcriptional regulators in bacteria?

While EF_2866 is specifically characterized as a transcriptional regulatory protein in Enterococcus faecalis, it shares functional similarities with other bacterial transcriptional regulators. Comparing EF_2866 to better-characterized transcriptional regulators can provide insights into its potential mechanisms of action.

For example, transcriptional regulators like SREBP-1 in other systems function by binding to specific DNA sequences in promoter regions. SREBP-1 binds to sterol regulatory element-1 (SRE1), a decamer sequence that influences the expression of various genes . Similarly, EF_2866 likely binds to specific DNA motifs to regulate gene expression in E. faecalis.

Many bacterial transcriptional regulators also share structural features such as the basic helix-loop-helix-leucine zipper (bHLH-Zip) domain found in SREBP-1 . Sequence analysis of EF_2866 could reveal similar DNA-binding domains that facilitate its interaction with target genes.

Comparative analyses should include:

• Sequence alignment with known transcriptional regulators

• Domain structure prediction

• Phylogenetic analysis to identify evolutionary relationships

• Functional characterization of DNA-binding specificity

How can recombinant EF_2866 be used as a vector for antigen delivery?

The potential application of recombinant E. faecalis strains as vectors for antigen delivery represents an innovative approach that could be extended to EF_2866. Drawing from research with similar E. faecalis systems, the following methodological framework can be outlined:

• Vector construction: Create a plasmid containing:

  • Signal peptide (SP) sequence for secretion

  • The antigen of interest

  • Cell wall anchor (CWA) sequence for surface display

  • Appropriate promoter and regulatory elements

• Transformation: Electroporate the constructed plasmid into a suitable E. faecalis strain (similar to the MDXEF-1 strain used in other studies)

• Verification of expression:

  • Confirm surface anchoring of the fusion protein using Western blot

  • Detect the expressed protein using appropriate antibodies

• Immunization protocol:

  • Administer the recombinant bacteria orally or via another appropriate route

  • Follow a multi-dose regimen (e.g., 3 immunizations at 2-week intervals)

• Immune response assessment:

  • Measure antibody responses (IgG in sera, secretory IgA)

  • Analyze cellular immunity (CD4+/CD8+ T cell proportions)

  • Quantify cytokine expression (particularly IL-2 and IFN-γ)

This approach has shown success with other E. faecalis strains, where fusion proteins incorporating dendritic cell-targeting peptides (DCpep) significantly enhanced immune responses. The DCpep functions by binding to receptors on dendritic cells, facilitating uptake and presentation of the fusion protein, which ultimately leads to T cell activation and enhanced immune responses .

What factors influence the expression and stability of EF_2866 in experimental systems?

Multiple factors can impact the expression and stability of recombinant EF_2866 in experimental systems. Based on studies with similar transcriptional regulatory proteins, the following factors should be considered:

Table 1: Factors Influencing EF_2866 Expression and Stability

Transcriptional regulators like EF_2866 often contain DNA-binding domains that can be sensitive to experimental conditions. Similar to observations with other transcription factors such as PRRX1, expression levels can be influenced by growth factors and cellular signaling pathways . For example, TGF-β1 treatment has been shown to decrease expression of certain transcription factors at both mRNA and protein levels, while other factors like prostaglandin E2 (PGE2) can increase expression .

What are the challenges in identifying the gene targets of EF_2866?

Identifying the specific gene targets regulated by EF_2866 presents several methodological challenges. Based on approaches used for other transcriptional regulators, the following comprehensive strategy can be implemented:

• Chromatin Immunoprecipitation followed by sequencing (ChIP-seq):

  • Generate specific antibodies against recombinant EF_2866

  • Cross-link protein-DNA complexes in vivo

  • Immunoprecipitate EF_2866-bound DNA fragments

  • Sequence and map binding sites to the E. faecalis genome

• Transcriptomic analysis:

  • Compare gene expression profiles between wild-type and EF_2866 knockout/overexpression strains

  • Conduct RNA-seq under various environmental conditions

  • Identify differentially expressed genes that correlate with EF_2866 levels

• Electrophoretic Mobility Shift Assays (EMSA):

  • Express and purify recombinant EF_2866

  • Generate labeled DNA probes from putative binding regions

  • Assess binding through gel shift analysis

  • Perform competition assays to confirm specificity

• Reporter gene assays:

  • Clone potential target promoters upstream of reporter genes

  • Co-express with EF_2866 in heterologous systems

  • Measure reporter activity to assess regulatory effects

Challenges in this process include distinguishing direct from indirect regulatory effects, identifying co-factors that may influence binding specificity, and accounting for context-dependent regulation that might occur only under specific environmental conditions. Additionally, the "probable" classification of EF_2866 suggests uncertainty about its exact function, requiring validation through multiple complementary approaches.

How do post-translational modifications affect EF_2866 function?

Post-translational modifications (PTMs) can significantly impact the function of transcriptional regulators like EF_2866. Though specific PTMs of EF_2866 are not detailed in the available search results, insights can be drawn from studies of similar regulatory proteins.

For transcriptional regulators, common PTMs include:

• Phosphorylation: Often regulates DNA-binding activity and protein-protein interactions

• Acetylation: Can affect nuclear localization and DNA-binding affinity

• SUMOylation: Frequently modulates transcriptional activity

• Ubiquitination: Controls protein stability and turnover

To investigate PTMs in EF_2866, researchers should employ the following methodological approaches:

• Mass spectrometry-based PTM mapping:

  • Digest purified EF_2866 with proteases (trypsin, chymotrypsin)

  • Analyze resulting peptides using high-resolution MS

  • Compare observed masses with theoretical masses to identify modifications

  • Use data-independent acquisition mass spectrometry (DIA-MS) with parameters including 1% FDR at spectral, peptide, and protein group levels

• Site-directed mutagenesis:

  • Identify potential modification sites through in silico analysis

  • Generate point mutations at these sites

  • Assess the functional impact on DNA binding and transcriptional activity

• Phosphorylation-specific analytical techniques:

  • Use Phos-tag SDS-PAGE to detect phosphorylated species

  • Employ phospho-specific antibodies for Western blotting

  • Conduct in vitro kinase assays to identify responsible kinases

• Functional correlation studies:

  • Compare PTM patterns under different environmental conditions

  • Correlate modifications with changes in transcriptional activity

  • Identify environmental triggers that induce specific modifications