Recombinant Clostridium kluyveri Elongation factor Ts (tsf)

What is the functional role of Elongation Factor Ts (tsf) in Clostridium kluyveri protein synthesis?

EF-Ts acts as a nucleotide exchange factor for EF-Tu, accelerating the replacement of GDP with GTP to reactivate EF-Tu for subsequent rounds of aminoacyl-tRNA delivery to the ribosome . In C. kluyveri, this process is critical for maintaining translation rates during rapid growth phases, particularly under substrate-rich conditions like those encountered in ethanol-based chain elongation environments . Methodologically, researchers can quantify EF-Ts activity using in vitro assays measuring GDP release rates from EF-Tu via stopped-flow fluorescence (using mant-GDP as a reporter) or nitrocellulose filter binding assays .

How should researchers design experiments for recombinant EF-Ts expression and purification?

The yeast-expressed recombinant EF-Ts (UniProt A5N829) requires careful handling due to its temperature sensitivity :

Table 1: Recombinant EF-Ts Expression Parameters

Critical considerations include:

• Tag positioning: N-terminal vs. C-terminal tags affect solvent accessibility and functional integrity

• Protease selection: Avoid thrombin if surface-exposed cleavage sites exist in the native sequence

What are common experimental artifacts when analyzing EF-Ts/EF-Tu interactions?

Three key artifacts require mitigation:

• Non-specific binding: Use high-salt buffers (≥150 mM NaCl) during pull-down assays to reduce ionic interactions

• GTP hydrolysis interference: Include non-hydrolyzable GTP analogs (GMPPNP) in complex stabilization

• Oxidative dimerization: Maintain 1-5 mM DTT in buffers to prevent cysteine-mediated aggregation

How can researchers resolve contradictions in EF-Ts kinetic data across studies?

Discrepancies in reported $K_d$ values (EF-Ts:EF-Tu binding) often stem from:

Table 2: Kinetic Variability Sources

A 2024 comparative study recommends:

• Parallel measurements using isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR)

• Normalization against a reference system (e.g., E. coli EF-Ts/EF-Tu)

What advanced structural methods elucidate EF-Ts mechanism in C. kluyveri?

Recent advances combine:

• Cryo-EM (2.8-Å resolution) to capture transient ternary complexes with EF-Tu and tRNA

• Hydrogen-deuterium exchange MS: Maps conformational changes during nucleotide exchange

• 19F-NMR: Probes real-time dynamics of critical residues (e.g., Asp154 in the GDP displacement loop)

A multi-technique approach revealed that C. kluyveri EF-Ts induces a 23° rotation in EF-Tu's Domain III compared to the E. coli homolog, explaining species-specific activity differences .

How does EF-Ts functionality integrate with C. kluyveri's metabolic pathways?

EF-Ts directly impacts chain elongation efficiency through:

• Translation rate modulation: Higher EF-Ts levels correlate with 2.3× increased n-caproate production under CO₂ supplementation

• Energy sensing: EF-Tu GTPase activity links amino acid availability to acetate/ethanol utilization via (p)ppGpp signaling

Integrated Experimental Design

• Cultivate C. kluyveri in controlled bioreactors with variable CO₂ (0-3 mL/min)

• Quantify EF-Ts expression via targeted proteomics

• Correlate with:

  • n-Caproate yields (GC-MS)

  • Translational fidelity (β-galactosidase reporter assays)

Addressing low recombinant EF-Ts stability in in vitro assays

Problem: Activity loss >40% after 4 hours at 25°C
Solutions:

• Add 0.01% Tween-20 to prevent surface adsorption

• Pre-incubate with 2 mM MgATP to stabilize tertiary structure

• Use capillary electrophoresis for rapid (<5 min) activity measurements

Reconciling in vitro vs. in vivo EF-Ts efficiency measurements

Discrepancies arise from compartmentalization effects. A 2024 in silico study recommends:

• Measure intracellular viscosity (FRAP with GFP-EF-Ts)

• Adjust in vitro assays with 15% Ficoll-400 to mimic cytoplasmic crowding

• Use single-molecule tracking in live cells for direct comparison

Can EF-Ts be engineered for improved translation in synthetic biology applications?

Recent attempts using ancestral sequence reconstruction yielded three variants with enhanced properties:

Table 3: Engineered EF-Ts Variants

Key methodology:

• Phylogenetic analysis of 87 Clostridial EF-Ts sequences

• Combinatorial site-saturation mutagenesis at conserved residues

How does EF-Ts interact with antibiotic resistance mechanisms?

EF-Ts overexpression partially compensates for kasugamycin-induced translational stalling in C. kluyveri:

• 72% survival at 50 μg/mL kasugamycin vs 22% in EF-Ts knockdown strains

• Mechanism involves enhanced ribosome recycling (18S rRNA protection assay)