Recombinant Sulfolobus solfataricus Elongation factor 2 (fusA), partial
Description
Production and Purification
Expression system:
| Parameter | Detail |
|---|---|
| Host | Escherichia coli BL21 (DE3) |
| Vector | pETM11+ or pRSETB |
| Induction | IPTG-driven T7 promoter |
| Purification | Ni-NTA affinity chromatography |
Thermal stability assays confirm functionality at 65°C, consistent with S. solfataricus’s optimal growth temperature . The absence of diphthamide simplifies in vitro studies but may alter interaction kinetics compared to native EF-2 .
GTPase Activity
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Demonstrated ribosome-dependent GTP hydrolysis (k~cat~ = 0.25 min⁻¹) .
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Activity inhibited by non-hydrolyzable GTP analogs (e.g., GMP-PNP) .
Role in Translation Initiation
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Induces release of archaeal Initiation Factor 6 (aIF6) from 50S ribosomal subunits, enabling translation elongation .
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Mechanism: GTP hydrolysis triggers conformational changes that displace aIF6 from its binding site near the ribosomal L1 stalk .
Key experimental results:
| Condition | aIF6 Release Efficiency |
|---|---|
| EF-2 + GTP | 95% ± 3% |
| EF-2 + GMP-PNP | <5% |
| EF-2 + 50S subunits | 90% ± 4% |
Interactions With Ribosome Maturation Factors
Competition with aSBDS:
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Archaeal SBDS (aSBDS) and EF-2 exhibit overlapping binding sites on the 50S subunit .
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Structural modeling predicts steric hindrance between aSBDS and EF-2, suggesting temporal regulation during ribosome assembly .
Functional implications:
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aSBDS may act as a placeholder during ribosomal biogenesis, delaying EF-2 binding until maturation is complete .
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EF-2’s role extends beyond translation elongation to ribosome quality control .
Technical Advancements Enabled by Recombinant EF-2
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Vector systems: Modified S. solfataricus expression vectors (e.g., pSVA series) with inducible promoters (tf55α, araS) improve yields of functional tagged proteins .
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Applications:
Limitations and Future Directions
Product Specs
Target Background
KEGG: sso:SSO0728
STRING: 273057.SSO0728
