Recombinant Bacillus cereus Elongation factor Ts (tsf)
Description
Molecular Definition and Significance
Recombinant B. cereus EF-Ts (tsf) is derived from the tsf gene (UniProt ID: Q812X3) and expressed in Escherichia coli systems . It functions as a guanine nucleotide exchange factor (GEF) for EF-Tu, accelerating the replacement of GDP with GTP to regenerate active EF-Tu- GTP complexes required for translation elongation .
Primary Structure
The recombinant protein comprises 178 amino acids with the following N-terminal sequence :
MAITAQMVKE LREKTGAGMM DCKKALTETN GDMEKAIDFL REKGIAKAAK KADRIAAEGL TFIETNGNDA LILELNSETD FVAKNEGFQT LIKELAAHLL TNKPANVEEA MAQTMENGKK VEEHINEAIA KIGEKLTLRR FEIVSKTDAD
Domain Organization
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EF-Ts binding domain: Directly interacts with EF-Tu to destabilize GDP binding .
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Catalytic motifs: Includes residues critical for nucleotide exchange activity, though B. cereus-specific motifs remain understudied compared to E. coli EF-Ts .
Functional Role in Translation
EF-Ts enhances the dynamics of EF-Tu- GTP- aa-tRNA ternary complex formation and dissociation through two mechanisms :
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Accelerated GDP release: Reduces EF-Tu’s affinity for GDP by 100-fold, enabling GTP binding.
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Conformational regulation: Stabilizes EF-Tu’s GTP-bound state, increasing ternary complex stability.
Key kinetic parameters (in vitro) :
| Process | Rate Constant (EF-Ts absent) | Rate Constant (EF-Ts present) |
|---|---|---|
| GDP release from EF-Tu | 0.02 s⁻¹ | 2.5 s⁻¹ |
| Ternary complex formation | 1.5 × 10⁴ M⁻¹s⁻¹ | 4.8 × 10⁵ M⁻¹s⁻¹ |
Expression System
Purity and Stability
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Storage: Lyophilized form stable for 12 months at -20°C/-80°C; liquid form stable for 6 months under the same conditions .
Thermostability
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Retains >90% activity after 1 hour at 50°C in phosphate buffer (pH 7.0) .
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Denatures above 60°C, as shown by circular dichroism (CD) spectra .
pH Tolerance
Research Applications
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Protein synthesis studies: Used to investigate EF-Tu’s conformational changes during translation .
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Chaperone interactions: Demonstrated to modulate Hsp33-mediated EF-Tu aggregation, linking translation fidelity to stress response .
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Antibiotic development: Potential target for disrupting bacterial protein synthesis .
Comparative Analysis with Homologs
| Property | B. cereus EF-Ts | E. coli EF-Ts |
|---|---|---|
| Molecular weight (kDa) | 20.5 | 19.8 |
| Optimal pH | 7.0 | 7.5 |
| Thermostability (θ½) | 50°C | 45°C |
| Binding affinity (Kd) | 4.2 nM (GDP) | 3.8 nM (GDP) |
Product Specs
Target Background
KEGG: bcq:BCQ_3611
