Recombinant Cell division protein SepF 3 (sepF3)

What is Cell division protein SepF 3 and what is its role in bacterial division?

SepF3 belongs to the SepF protein family, which functions as membrane anchors for the Z ring during bacterial cell division. SepF proteins form large protein rings that serve as a scaffold for the assembly of the division machinery. Research has shown that SepF functions as a membrane anchor for the Z ring, similar to FtsA in some bacterial species . This anchoring function is critical for proper Z-ring formation and subsequent cell division processes. The membrane-binding domain of SepF is typically located at the very beginning of the protein, which enables it to interact with the cell membrane while simultaneously binding to FtsZ filaments .

How does SepF3 interact with other cell division proteins?

SepF3 interacts with multiple cell division proteins, most notably FtsZ, the primary component of the bacterial Z ring. Studies have demonstrated that SepF proteins interact with FtsZ and MurG to regulate cell growth and division . The interaction with FtsZ is particularly important as it enables the anchoring of FtsZ polymers to the cell membrane, creating the characteristic ring-like structure that defines the division site. The SepF-FtsZ interaction involves the FtsZ-binding domain of SepF, which has been structurally characterized . Additionally, SepF's interaction with MurG suggests a role in coordinating cell wall synthesis with the division process, highlighting its multifunctional nature in bacterial cell division.

Why is SepF essential in certain bacterial species but not others?

SepF is particularly essential in bacterial species that lack FtsA, another protein capable of anchoring the Z ring to the cell membrane . In mycobacteria, for example, SepF plays an essential role in cell division . This differential requirement across bacterial species reflects evolutionary adaptations in cell division mechanisms. In species where both FtsA and SepF are present, they may have partially redundant functions, allowing the organism to maintain division capability even if one system is compromised. The molecular basis for this complementarity involves their shared ability to bind both FtsZ and the cell membrane, though through different structural mechanisms.

What expression systems are optimal for recombinant SepF3 production?

For recombinant SepF3 production, E. coli-based expression systems are generally recommended. While traditional T7 RNA polymerase-dependent expression systems (like BL21(DE3)) can be effective, they often lack fine control over expression levels. The RiboTite gene expression control system represents an advanced alternative that allows precise tuning of expression levels to match secretion capacity . This system has demonstrated absolute control of basal expression in the absence of induction and excellent dynamic range control of gene expression and secretion, with regression coefficients showing excellent linearity . For periplasmic expression, proper signal peptide selection is crucial, with options like DsbA-E1 and PiiiE5 showing variable performance depending on culture conditions.

How can expression conditions be optimized to maximize SepF3 yield and quality?

Optimization of SepF3 expression requires balancing multiple parameters:

• Temperature control: Lower induction temperatures (typically 30°C) often improve protein folding and solubility compared to standard 37°C cultivation .

• Signal peptide selection: Different signal peptides can significantly affect both expression and secretion efficiency. Experimental data shows secretion efficiencies ranging from 16-30% depending on the signal peptide used .

• Expression system selection: The RiboTite system has demonstrated up to ninefold enhanced secretion titer compared to classical T7 RNAP-dependent systems .

• Cultivation method: Fed-batch fermentation conditions can significantly improve yields compared to batch shake flask conditions, with some expression differences between signal peptides being reduced under fed-batch conditions .

• Induction strategy: Fine-tuning inducer concentration allows matching expression levels to the cell's secretion capacity, preventing toxic accumulation of unfolded proteins.

What analytical methods should be employed to verify the quality of purified recombinant SepF3?

Quality assessment of recombinant SepF3 should include:

• Purity analysis using SDS-PAGE and western blotting to confirm identity and absence of degradation products.

• Mass spectrometry analysis to verify protein mass and detect any post-translational modifications. LC-MS/MS data can be analyzed via search engines like MaxQuant with parameters including oxidation (M) and protein N-terminal acetylation (N) as variable modifications and carbamidomethylation of cysteine as a fixed modification .

• Structure verification through circular dichroism to confirm proper secondary structure formation.

• Functional validation through FtsZ binding assays and membrane interaction studies to confirm biological activity.

• Size exclusion chromatography to verify the formation of characteristic SepF rings, which are critical for its function.

What are the recommended experimental approaches for studying SepF3-FtsZ interactions?

For investigating SepF3-FtsZ interactions, researchers should consider:

• In vitro reconstitution assays using purified components to directly visualize interaction effects on FtsZ polymerization.

• Co-sedimentation assays to quantify the binding of SepF3 to FtsZ polymers under various conditions.

• Surface plasmon resonance (SPR) to determine binding kinetics and affinity constants.

• Cryo-electron microscopy to visualize the structural basis of SepF rings interacting with FtsZ filaments.

• Fluorescence microscopy with labeled proteins to track interactions in real-time.

• Mutational analysis targeting the FtsZ-binding domain of SepF3 to identify critical residues for interaction.

How can single-case experimental designs be applied to SepF3 research?

Single-case experimental designs (SCEDs) can be effectively adapted for SepF3 research:

• Reversal designs can test the effects of adding and removing SepF3 on FtsZ assembly or cell division processes. This provides multiple demonstrations of treatment effects: B1 to C1 (addition), C1 to B2 (removal), and B2 to C2 (readdition) .

• Multiple baseline designs allow evaluation of SepF3 function across different experimental conditions or bacterial species.

• Combined multiple baseline/reversal designs can identify optimal conditions for SepF3 activity or expression.

These designs are particularly valuable for treatment development and personalized interventions, offering a flexible and cost-effective approach to determining optimal experimental conditions . To enhance validity, researchers can randomize the order of experimental conditions and implement blinding in intervention and data collection phases when possible.

What proteomics approaches are most effective for studying SepF3 interactions?

Effective proteomics approaches for SepF3 interaction studies include:

• Label-free quantitative (LFQ) proteomics with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify protein-protein interactions.

• Sample preparation protocols should include at least three biological replicates and three technical replicates to ensure statistical robustness .

• Data analysis through MaxQuant search engine (version 1.6.0.16 or newer) against appropriate protein databases, with false discovery rates (FDRs) of peptide and protein set at 0.01 .

• Statistical analysis using two-sample t-tests based on normalized intensity values, with absent proteins identified from pre-imputation datasets .

• For pathway analysis, GO and KEGG analyses can be carried out with Z-scores used to count the enrichment values of the pathways .

How can structural biology techniques be applied to understand SepF3 function?

Advanced structural biology approaches provide critical insights into SepF3:

• X-ray crystallography can determine the atomic-level structure of SepF3's FtsZ-binding and ring-forming domains, as has been done for other SepF proteins .

• Cryo-electron microscopy is particularly valuable for visualizing SepF3 rings and their interaction with FtsZ filaments.

• Nuclear magnetic resonance (NMR) spectroscopy can characterize the dynamics of SepF3-membrane interactions.

• Molecular dynamics simulations can predict how SepF3's membrane-binding domain interacts with lipid bilayers.

• Small-angle X-ray scattering (SAXS) can provide information about SepF3 ring formation in solution under different conditions.

These approaches collectively reveal how SepF3's structure enables its dual function of membrane binding and FtsZ interaction, explaining its ability to anchor the Z ring to the cell membrane similarly to FtsA .

What are the most effective approaches for studying SepF3-mediated membrane interactions?

For investigating SepF3-membrane interactions, researchers should consider:

• Liposome binding assays with defined lipid compositions to determine lipid preferences of SepF3.

• Fluorescence microscopy using labeled SepF3 to visualize membrane localization in bacterial cells.

• Atomic force microscopy to examine the topography of SepF3 rings on membrane surfaces.

• FRET-based assays to measure real-time interactions between SepF3 and membrane components.

• Site-directed mutagenesis targeting the membrane-binding domain located at the N-terminus of SepF3 .

These approaches can reveal how SepF3 associates with the bacterial membrane, providing insights into its functional role as a membrane anchor for the Z ring during bacterial cell division.

How can SepF3 research contribute to antibiotic development strategies?

SepF3 research offers promising avenues for antibiotic development:

• As SepF is essential in mycobacteria and other bacteria that lack FtsA , it represents a potential target for species-specific antibiotics.

• Structural characterization of the FtsZ-binding domain of SepF3 enables rational design of inhibitors that could disrupt Z-ring formation.

• The membrane-binding domain offers another potential target, as disrupting SepF3-membrane interactions would prevent Z-ring anchoring.

• High-throughput screening assays can be developed to identify compounds that inhibit SepF3 ring formation or its interaction with FtsZ.

• Bacterial strains with temperature-sensitive SepF mutations can serve as tools for validating potential inhibitors in vivo.

This research could lead to novel antibiotics with unique mechanisms of action, potentially addressing the growing problem of antibiotic resistance by targeting previously unexploited cellular components.

What strategies can address solubility issues with recombinant SepF3?

Addressing solubility challenges with recombinant SepF3 requires multiple approaches:

• Expression temperature optimization: Lower temperatures (30°C) during induction can significantly improve solubility by slowing protein synthesis and allowing proper folding .

• Fusion tag selection: Solubility-enhancing tags such as MBP (maltose-binding protein) or SUMO can improve folding and prevent aggregation.

• Buffer optimization: Screening different buffer compositions, pH values, and salt concentrations can identify conditions that maximize SepF3 solubility.

• Co-expression with chaperones: Molecular chaperones can assist proper folding of SepF3 during expression.

• Periplasmic secretion: Directing SepF3 to the periplasm using appropriate signal peptides can improve folding, with secretion efficiencies ranging from 16-30% depending on the signal peptide used .

• Expression level control: Fine-tuning expression using systems like RiboTite prevents overwhelming the cell's folding machinery, maintaining better balance between expression and proper folding .

How can researchers optimize periplasmic secretion of SepF3?

Optimizing periplasmic secretion of SepF3 requires:

• Signal peptide selection: Different signal peptides (e.g., DsbA-E1, Piii-E5) show variable performance in directing SepF3 to the periplasm, with secretion efficiencies ranging from 16-30% .

• Secretion pathway consideration: SepF3 may be better suited for either the SecB-dependent (post-translational) or SRP-dependent (co-translational) secretion pathway, with research indicating better coordination between expression and secretion for the SRP-dependent pathway .

• Expression control: Using the RiboTite system allows fine-tuning of expression levels to match secretion capacity, preventing toxic accumulation of unfolded protein in the cytoplasm .

• Culture conditions: Fed-batch fermentation can significantly improve periplasmic yields compared to batch shake flask conditions, potentially reducing performance differences between signal peptides .

• Temperature management: Lower induction temperatures (30°C) promote proper folding during translocation to the periplasm .

These approaches collectively ensure that SepF3 expression is properly coordinated with secretion capacity, maximizing yield and quality of the recombinant protein.

What controls should be included in SepF3 functional studies?

Rigorous functional studies of SepF3 require comprehensive controls:

• Negative controls: FtsZ alone without SepF3 to establish baseline polymerization or membrane binding behavior.

• Positive controls: Known FtsZ-binding proteins (like FtsA) to validate assay functionality and provide comparison points.

• Mutant SepF3 variants: Proteins with mutations in key functional domains (FtsZ-binding, membrane-binding, or ring-forming) to confirm specificity.

• Denatured SepF3: Heat-inactivated or chemically denatured protein to confirm that native structure is required for observed activities.

• Concentration gradients: Testing multiple concentrations of SepF3 to establish dose-response relationships.

• Heterologous proteins: Structurally similar but functionally distinct proteins to confirm specificity of observed effects.

These controls ensure that experimental observations genuinely reflect SepF3's specific biological functions rather than experimental artifacts or non-specific effects.