Recombinant Rhodococcus opacus Elongation factor Ts (tsf)

How does Elongation Factor Ts expression differ under various metabolic conditions in Rhodococcus opacus?

Expression of translational machinery proteins like EF-Ts in R. opacus can vary depending on growth conditions and carbon sources. Transcriptomic analyses reveal that when R. opacus grows on aromatic compounds like phenol, significant metabolic reprogramming occurs, potentially affecting expression of central metabolic and protein synthesis genes . The expression patterns of translation factors may correlate with growth rates, which are demonstrably different when R. opacus is grown on various carbon sources.

What are the recommended protocols for expression and purification of recombinant Elongation Factor Ts?

Based on established protocols for similar bacterial proteins, recombinant Elongation Factor Ts from R. opacus can be expressed in E. coli expression systems . The following methodology is recommended:

• Cloning: The tsf gene should be PCR-amplified from R. opacus genomic DNA and cloned into an appropriate expression vector with a suitable tag (determined during manufacturing).

• Expression conditions: Transform the construct into an E. coli expression strain and induce protein expression under optimized conditions (typically IPTG induction at mid-log phase, followed by growth at lower temperatures, e.g., 18-25°C for 12-16 hours).

• Purification: Employ affinity chromatography based on the fusion tag, followed by size exclusion chromatography to achieve >85% purity as verified by SDS-PAGE .

• Quality control: Verify protein identity through mass spectrometry and assess activity through GDP/GTP exchange assays.

How should researchers handle and store recombinant Elongation Factor Ts to maintain optimal stability and activity?

To maximize stability and activity of recombinant EF-Ts from R. opacus:

• Reconstitution: Before opening the vial containing lyophilized protein, centrifuge briefly to ensure all content settles at the bottom. Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• Storage conditions:

  • For long-term storage: Add glycerol to a final concentration of 5-50% (recommended default is 50%) and store in aliquots at -20°C or -80°C .

  • For working stocks: Store aliquots at 4°C for up to one week .

  • Avoid repeated freeze-thaw cycles as this significantly reduces protein activity .

• Shelf life considerations:

  • Liquid preparations typically maintain activity for approximately 6 months at -20°C/-80°C.

  • Lyophilized preparations generally have a shelf life of 12 months at -20°C/-80°C .

The stability of the protein is influenced by multiple factors including buffer composition, storage temperature, and inherent stability of the protein itself .

How can Elongation Factor Ts be integrated into genome-scale models of Rhodococcus opacus metabolism?

Integrating EF-Ts into genome-scale models (GSMs) of R. opacus metabolism requires:

• Gene annotation: Identify and annotate the tsf gene within the R. opacus genome. In the current genome-scale model iGR1773, which includes 1773 genes, the translation machinery components should be properly represented .

• Reaction inclusion: Incorporate reactions representing EF-Ts activity in protein synthesis, including:

  • GDP/GTP exchange reactions

  • EF-Tu·EF-Ts complex formation and dissociation

  • ATP consumption associated with translation elongation

• Flux constraints: Apply appropriate constraints to translation-related reactions based on experimental data. The E-Flux2 method, which has been shown to provide better predictions of R. opacus metabolism than standard FBA methods, can be used to incorporate transcriptomic data on tsf expression into the model .

• Validation: Validate model predictions by comparing growth rates under different conditions with experimental data. For R. opacus, E-Flux2 has demonstrated superior performance in predicting both growth rates and central carbon fluxes (R² = 0.96 for phenol metabolism) .

What role might Elongation Factor Ts play in the adaptation of Rhodococcus opacus to aromatic compound metabolism?

The adaptation of R. opacus to growth on aromatic compounds involves complex transcriptional reprogramming . Elongation Factor Ts may contribute to this adaptation through:

What are common issues encountered when working with recombinant Elongation Factor Ts and how can they be addressed?

Researchers commonly encounter these challenges when working with recombinant EF-Ts:

• Protein insolubility:

  • Problem: EF-Ts may form inclusion bodies during overexpression.

  • Solution: Optimize expression conditions by lowering induction temperature (16-20°C), reducing inducer concentration, or using solubility-enhancing fusion tags.

• Loss of activity upon purification:

  • Problem: Purified EF-Ts may show reduced nucleotide exchange activity.

  • Solution: Include stabilizing agents (glycerol, reducing agents) in purification buffers and minimize exposure to extreme temperatures or pH conditions.

• Degradation during storage:

  • Problem: Protein degradation during storage reduces experimental reproducibility.

  • Solution: Store with recommended glycerol concentration (5-50%), aliquot to avoid freeze-thaw cycles, and maintain at appropriate temperatures (-20°C/-80°C for long-term storage) .

• Activity assay challenges:

  • Problem: Difficulties in establishing reliable activity assays for EF-Ts.

  • Solution: Implement coupled assays that monitor nucleotide exchange on EF-Tu, using either fluorescent nucleotide analogs or mant-GDP/GTP for real-time measurements.

How can researchers overcome challenges in studying EF-Ts interactions with other components of the translation machinery?

Studying EF-Ts interactions with translation components presents several challenges:

• Complex formation analysis:

  • Challenge: Detecting transient interactions between EF-Ts and EF-Tu or other factors.

  • Approach: Employ surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or microscale thermophoresis (MST) for quantitative binding analysis under various conditions.

• Functional reconstitution:

  • Challenge: Reconstituting translation elongation with purified components.

  • Approach: Establish in vitro translation systems using purified ribosomes, translation factors, and mRNA templates to evaluate EF-Ts contribution to translation efficiency.

• Structural determination:

  • Challenge: Obtaining structural information of EF-Ts alone or in complexes.

  • Approach: Combine X-ray crystallography, cryo-EM, and molecular dynamics simulations to elucidate structural features and conformational changes.

• Integration with metabolic states:

  • Challenge: Correlating EF-Ts activity with metabolic conditions in R. opacus.

  • Approach: Combine transcriptomics, proteomics, and metabolic flux analysis (13C-MFA) to establish relationships between translation factor activity and metabolic states when growing on different carbon sources .

What techniques are most effective for studying the impact of EF-Ts on translation efficiency in the context of aromatic compound metabolism?

To investigate EF-Ts influence on translation during aromatic compound metabolism, researchers should consider: