Recombinant Anaerocellum thermophilum Elongation factor Ts (tsf)

Definition and Canonical Role of EF-Ts

Elongation Factor Ts (EF-Ts) facilitates the regeneration of elongation factor Tu (EF-Tu) by catalyzing GDP-to-GTP exchange, enabling EF-Tu to resume its role in delivering aminoacyl-tRNA to ribosomes during translation . In thermophiles like T. thermophilus, EF-Ts forms a stable homodimer with a disulfide bridge (Cys190) and retains activity at high temperatures .

Expression Systems

• Host Organism: Escherichia coli BL21(DE3)pLysS is commonly used for heterologous expression due to efficient plasmid uptake and protein yield .

• Cloning: The tsf gene (encoding EF-Ts) is cloned into expression vectors like pCold-1 (Takara) with N-terminal His-tags for purification .

• Induction: Protein expression is induced with IPTG (0.5–1 mM) at low temperatures (15°C) to enhance solubility .

Purification and Stability

• Thermostability: T. thermophilus EF-Ts withstands high-temperature purification steps (e.g., heat treatment at 70°C), enabling selective removal of E. coli contaminants .

• Dimerization: Purified EF-Ts forms homodimers stabilized by disulfide bonds, though dimerization is not essential for nucleotide exchange activity .

Key Domains and Motifs

Functional Interactions

• EF-Tu Binding: EF-Ts binds EF-Tu in a 1:1 ratio to mediate GDP release .

• Chaperone Synergy: Trigger factor (TF) and Hsp33 modulate EF-Tu stability, indirectly influencing EF-Ts activity under stress .

• Thermal Adaptation: Unique loops in thermophilic EF-Ts (e.g., residues 182–191 in T. aquaticus) enhance ribosome interaction and thermostability .

Comparative Analysis with Mesophilic Homologs

Industrial Enzymology

• Thermostable Tool: Recombinant EF-Ts from thermophiles is used in high-temperature enzymatic assays and synthetic biology platforms .

• Antibiotic Targets: EF-Ts’ interaction with EF-Tu is explored for developing elfamycin derivatives, though solubility challenges persist .

Research Tools

• Crystallography: T. thermophilus EF-Ts serves as a model for studying nucleotide exchange mechanisms due to its structural rigidity .

• Mutagenesis Studies: Truncated variants (e.g., trypsin-cleaved EF-Ts after Lys48) help map functional domains .

Unresolved Questions and Future Directions

• Disulfide Bridge Role: The physiological relevance of the Cys190 bond in T. thermophilus EF-Ts remains unclear .

• Moonlighting Functions: EF-Ts may have secondary roles in stress response or biofilm formation, as seen in EF-Tu homologs .

• Engineering: Optimizing expression systems for A. thermophilum EF-Ts could enhance its utility in industrial processes.