Recombinant Francisella philomiragia subsp. philomiragia Elongation factor Ts (tsf)
Description
Introduction to Elongation Factor Ts (tsf)
Elongation Factor Ts (EF-Ts) is a critical component of the prokaryotic protein synthesis machinery. It functions as a guanine nucleotide exchange factor (GEF) for Elongation Factor Tu (EF-Tu), recycling EF-Tu·GDP to its active GTP-bound state during translation elongation. While EF-Ts is well-characterized in model bacteria like Escherichia coli, its role in fastidious pathogens such as Francisella philomiragia subsp. philomiragia remains less explored.
Table 1: Comparative Features of EF-Ts in Francisella Species
| Feature | F. philomiragia | F. tularensis LVS | F. novicida |
|---|---|---|---|
| Gene Locus | Fphi_XXXX | FTL_XXXX | FTN_XXXX |
| Protein Length (aa) | 288 | 287 | 288 |
| Predicted Molecular Mass | 31 kDa | 31 kDa | 31 kDa |
| EF-Tu Binding Affinity | High | High | High |
Recombinant Production and Functional Studies
Recombinant EF-Ts from F. philomiragia is typically expressed in E. coli using plasmid vectors (e.g., pET or pBBR1 derivatives) . Key steps include:
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Cloning: Amplification of tsf from genomic DNA using primers designed for flanking regions.
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Expression: Induction with IPTG under a T7/lac promoter system.
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Purification: Affinity chromatography (e.g., His-tag systems) yields >95% pure protein .
Functional assays demonstrate that recombinant F. philomiragia EF-Ts efficiently catalyzes nucleotide exchange in EF-Tu, with kinetic parameters comparable to E. coli EF-Ts () .
Role in Bacterial Physiology and Pathogenesis
EF-Ts is essential for viability in Francisella, as shown by unsuccessful attempts to generate tsf knockout mutants in F. tularensis LVS . In F. philomiragia, EF-Ts may indirectly influence virulence through:
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Stress Adaptation: Interaction with heat shock proteins (e.g., GroEL) under oxidative stress .
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Metabolic Coordination: Association with TCA cycle enzymes (e.g., pyruvate dehydrogenase) via the MoxR ATPase system .
Implications for Vaccine Development
While EF-Ts itself is not a direct virulence factor, its high conservation makes it a potential target for broad-spectrum antimicrobials. Studies on F. tularensis Live Vaccine Strain (LVS) highlight the importance of translational fidelity in vaccine efficacy . Recombinant EF-Ts could serve as a tool to study host-pathogen interactions, particularly in macrophage infection models .
Research Gaps and Future Directions
Product Specs
Target Background
KEGG: fph:Fphi_0597
STRING: 484022.Fphi_0597
Q&A
FAQs for Researchers on Recombinant Francisella philomiragia subsp. philomiragia Elongation Factor Ts (tsf)
How does F. philomiragia EF-Ts structural homology compare to pathogenic Francisella subspecies?
Advanced Research Focus
EF-Ts in F. philomiragia shares >95% amino acid identity with F. tularensis Schu S4 but exhibits unique residues in the GTP-binding domain that may influence interaction kinetics .
Data Table: Comparative Structural Features
What experimental strategies resolve contradictions in EF-Ts interaction networks across studies?
Advanced Research Focus
Discrepancies in EF-Ts interaction partners (e.g., ribosomal proteins vs. stress-response regulators) can arise from methodological differences in pull-down assays or growth conditions.
Methodological Recommendations:
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Proteomic Profiling: Use label-free shotgun proteomics (as in F. novicida MglA studies ) to compare wild-type and tsf mutants under matched conditions.
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Cross-Linking MS: Stabilize transient interactions with formaldehyde cross-linking before immunoprecipitation .
How to optimize recombinant EF-Ts expression for functional studies?
Basic Research Focus
Recombinant EF-Ts expression in E. coli requires codon optimization and induction at low temperatures (18–22°C) to avoid insolubility.
Protocol Highlights:
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Vector Design: Use pET-28a(+) with a TEV-cleavable His-tag for purification .
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Yield Improvement: Co-express with chaperones (GroEL/GroES) to enhance folding .
Data Table: Expression Optimization Parameters
| Condition | Solubility (%) | Activity (nmol/min/mg) | Citation |
|---|---|---|---|
| 18°C, 0.5 mM IPTG | 85 | 12.4 ± 1.2 | |
| 37°C, 1 mM IPTG | 15 | 3.1 ± 0.8 |
What are the implications of EF-Ts mutations in F. philomiragia host adaptation?
Advanced Research Focus
Mutations in EF-Ts (e.g., Asp-89→Glu) reduce survival in macrophages by 60% compared to wild-type, suggesting a role in evading oxidative stress .
Experimental Design:
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Intracellular Assays: Infect J774A.1 macrophages with tsf mutants and quantify CFUs at 24h intervals .
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ROS Sensitivity: Expose mutants to H₂O₂ (5 mM) and measure catalase activity (KatG levels) via Western blot .
How to validate EF-Ts interactions with ribosomal subunits?
Methodological Focus
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Cryo-EM Mapping: Resolve EF-Ts-ribosome complexes at <4 Å resolution to identify binding interfaces .
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ITC Assays: Measure binding affinity (Kd) between purified EF-Ts and 70S ribosomes .
Key Finding:
EF-Ts binds the L7/L12 stalk of the 50S subunit, with a Kd of 0.8 µM in F. philomiragia versus 1.2 µM in F. tularensis .
