Recombinant Geobacillus stearothermophilus Translation initiation factor IF-2 (infB), partial
Description
Overview
Recombinant Geobacillus stearothermophilus Translation Initiation Factor IF-2 (infB), partial, is a genetically engineered fragment of the IF-2 protein, a critical GTPase involved in bacterial translation initiation. IF-2 facilitates ribosomal subunit association, initiator tRNA (fMet-tRNA) binding to the 30S ribosomal P-site, and GTP hydrolysis during the formation of the 70S initiation complex . The "partial" designation indicates the recombinant protein represents a functional domain rather than the full-length IF-2.
Functional Roles
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GTPase Activity: Ribosome-dependent GTP hydrolysis (Km for GTP: ~2-fold higher in the isolated G-domain vs. full IF-2; Vmax remains comparable) .
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Ribosome Binding: G-domain binds 50S subunits, while C2 weakly associates with 30S subunits .
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fMet-tRNA Stabilization: The C2 domain ensures proper tRNA orientation for initiation codon recognition .
Expression Systems
Example Protocol (Hypothetical)
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Gene Cloning: Amplify infB fragment (e.g., G2-G3-C1 domains) using primers with restriction sites (e.g., PmlI/XbaI) .
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Vector Construction: Subclone into expression vectors (e.g., pET-32b for E. coli) .
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Induction: Optimize with methanol (1% v/v) for P. pastoris or IPTG for E. coli .
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Purification: Ni-NTA chromatography for His-tagged variants .
Domain-Specific Activities
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G-Domain: Retains ribosome-dependent GTPase activity but shows reduced tRNA binding .
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C2 Domain: Degrades bacterial capsules (e.g., G. stearothermophilus) and inhibits biofilm formation .
Enzymatic Properties
| Parameter | G-Domain | Full-Length IF-2 |
|---|---|---|
| GTPase Km | 2× higher | Baseline |
| Thermostability | Stable up to 70°C | Less stable |
Applications
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Biotechnological: Engineered IF-2 domains are used to study translation mechanisms in thermophiles .
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Industrial: Thermostable IF-2 fragments aid in optimizing high-temperature enzymatic processes .
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Antimicrobial: C2-derived depolymerases target G. stearothermophilus biofilms in food spoilage .
