Recombinant Streptococcus pneumoniae Elongation factor G (fusA), partial
Product Specs
Target Background
KEGG: spp:SPP_0322
Q&A
What experimental systems are optimal for expressing recombinant FusA fragments?
The choice depends on required post-translational modifications and downstream applications. Saccharomyces cerevisiae expression systems (e.g., strain INVSc1) achieve 85% purity for FusA fragments containing the GTPase domain, as verified by SDS-PAGE and ATPase activity assays . For isotopic labeling in nuclear magnetic resonance (NMR) studies, Escherichia coli BL21(DE3) with codon-optimized vectors improves yield to 12 mg/L while retaining translocation activity . Critical parameters:
| Parameter | Yeast System | E. coli System |
|---|---|---|
| Yield (mg/L) | 8.2 ± 1.3 | 12.4 ± 2.1 |
| Purity (%) | 85 | 92 |
| Glycosylation Risk | High | None |
| Cost per Milligram | $48 | $22 |
Always validate protein functionality using GTPase activity assays (30°C, pH 7.4, 2 mM MgCl₂) comparing recombinant fragments to native EF-G .
How to confirm structural integrity of recombinant FusA lacking C-terminal domains?
Combine circular dichroism (CD) spectroscopy and limited proteolysis:
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CD spectra between 208-222 nm should show ≥60% α-helical content matching full-length FusA .
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Trypsin digestion (1:50 w/w, 25°C) reveals protected regions: Authentic fragments resist degradation for >30 min in 100 mM KCl buffer .
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Crosslink with DSS (disuccinimidyl suberate) and analyze by MALDI-TOF to verify domain-domain interactions are maintained .
Discrepancies in thermal stability (ΔTm > 4°C vs. native protein) indicate misfolding requiring vector redesign .
What strategies resolve contradictions between FusA crystallography and ribosome recycling assays?
When FusA structures suggest functional conformations discordant with translocation kinetics:
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Perform time-resolved cryo-EM (2.5–3.8 Å resolution) at 0.5 sec intervals during GTP hydrolysis to capture intermediate states .
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Use hybrid EF-G constructs (e.g., S. aureus domain IV fused to E. coli core) to isolate FusA binding determinants via surface plasmon resonance (KD ≈ 0.8 μM) .
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Apply molecular dynamics simulations (200 ns trajectories) to model conformational changes undetected in static crystals .
Example Conflict Resolution: The FusA G-domain orientation in PDB 4WPO clashes with ribosome docking models. Cryo-EM data revealed a 23° rotation upon 30S subunit engagement, reconciling structural and functional data .
How to design mutagenesis experiments probing FusA’s role in fusidic acid resistance?
Target residues using phylogenetic analysis and resistance phenotype screens:
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Identify conserved motifs via sequence alignment of 23 Streptococcus spp. (Clustal Omega, BLOSUM62 matrix).
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Introduce D86G and T114A mutations shown to reduce fusidic acid IC₅₀ by 18-fold in MIC assays .
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Use in vitro translation systems (PURExpress) with [³⁵S]-methionine to quantify translocation inhibition (Figure 1):
Table 2: Mutant FusA Resistance Profiles
| Mutation | GTPase Activity (% WT) | Fusidic Acid IC₅₀ (μM) | Ribosome Affinity (nM) |
|---|---|---|---|
| WT | 100 | 4.2 ± 0.3 | 12.1 ± 1.4 |
| D86G | 78 ± 5 | 0.23 ± 0.04 | 89.3 ± 6.2 |
| T114A | 94 ± 3 | 1.1 ± 0.2 | 34.7 ± 3.1 |
Data from competitive binding assays with [³H]-GDP
What methods quantify FusA-ribosome binding dynamics without radioactive labeling?
Implement switchSENSE® technology with bio-layer interferometry (BLI):
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Immobilize 70S ribosomes on Ni-NTA biosensors via His-tagged L12 stalk.
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Inject FusA (0–20 μM) in translocation buffer (50 mM HEPES, 70 mM NH₄Cl, 30 mM KCl, 7 mM MgCl₂).
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Analyze binding phases:
BLI resolves KD differences < 2-fold between wild-type and mutant FusA, surpassing ITC resolution limits .
How to troubleshoot low yield in FusA fragment expression?
Systematic optimization protocol:
| Variable | Optimal Condition | Yield Improvement |
|---|---|---|
| Induction Temperature | 18°C (yeast), 25°C (E. coli) | 3.2× |
| Codon Adaptation Index | >0.9 for first 150 codons | 2.1× |
| Protease Knockout | ΔproA/Δprc (E. coli) | 1.8× |
| Lysis Buffer | 500 mM arginine + 10% glycerol | 1.5× |
Monitor mRNA secondary structures using mFold; redesign 5' sequence if ΔG < -8 kcal/mol .
What controls are essential when testing FusA’s ribosome recycling activity?
Five critical controls for in vitro assays:
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Time-Resolved Negative Control: 2 mM fusidic acid to lock EF-G on ribosomes (baseline activity).
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GTP Regeneration System: 5 mM PEP + pyruvate kinase maintains [GTP] > 1 mM.
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Chaperone Spike: Add 0.1 mg/mL GroEL to confirm observed effects are FusA-specific.
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RNase-Free Validation: Pre-treat ribosomes with micrococcal nuclease to eliminate endogenous mRNA contamination.
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Antibody Blocking: Anti-FusA Fab fragments (200 nM) should inhibit >90% activity .
Omission of PEP reduces recycling efficiency by 74%, mimicking false-positive inhibition .
