Recombinant Francisella tularensis subsp. novicida Ribosome-recycling factor (frr)
Description
Recombinant Production and Purification
rFrr is typically produced in E. coli expression systems. A validated protocol includes:
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Cloning: The frr gene is amplified via PCR and cloned into a plasmid (e.g., pET-28a) with a 6xHis tag for affinity purification .
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Expression: Induction with IPTG at 18°C for 16 hours minimizes inclusion body formation.
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Purification: Nickel-affinity chromatography followed by size-exclusion chromatography yields >95% pure protein .
Table 2: Expression and Purification Metrics for rFrr
| Parameter | Value |
|---|---|
| Host organism | E. coli BL21(DE3) |
| Expression vector | pET-28a(+) |
| Yield | 12–15 mg/L culture |
| Purity | >95% (SDS-PAGE) |
| Oligomeric state | Monomeric (SEC-MALS) |
Functional Insights
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Ribosome recycling: rFrr dissociates 70S ribosomes into 50S and 30S subunits in vitro, with activity comparable to E. coli Frr .
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Thermostability: Differential scanning calorimetry (DSC) shows a melting temperature (Tm) of 58°C, indicating moderate stability .
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Interactome: Co-purification studies identify interactions with elongation factor G (EF-G) and ribosomal proteins L9 and S12 .
Applications in Research
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Antibiotic target validation: Frr is essential for bacterial survival, making it a candidate for novel antimicrobials .
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Structural studies: Crystallization trials of rFrr are ongoing to resolve its binding interface with the ribosome.
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Vaccine development: While not directly tested, Frr’s conservation across Francisella subspecies suggests potential as a cross-protective antigen .
Unresolved Questions
Product Specs
Target Background
KEGG: ftn:FTN_0230
