Recombinant Salmonella schwarzengrund Elongation factor Ts (tsf)

How does Recombinant Salmonella schwarzengrund Elongation factor Ts differ from native Elongation factor Ts?

Recombinant Salmonella schwarzengrund Elongation factor Ts is produced using E. coli expression systems rather than being isolated directly from Salmonella schwarzengrund cultures . Several key differences exist between recombinant and native forms:

• Affinity tags: Recombinant proteins typically contain affinity tags to facilitate purification. According to product information, "Tag type will be determined during the manufacturing process" .

• Post-translational modifications: Native proteins may contain modifications that are absent in recombinant versions expressed in heterologous systems.

• Folding characteristics: Expression conditions can affect protein folding, potentially resulting in conformational differences compared to the native form.

• Purity: Recombinant preparations generally achieve higher purity (>85% by SDS-PAGE as reported) than would be practical from native sources.

• Stability characteristics: Recombinant proteins may exhibit different stability profiles requiring specific storage conditions (-20°C or -80°C for extended storage) .

These differences should be considered when designing experiments, particularly for structural or functional studies where native conformation is critical.

What experimental approaches can be used to validate the functionality of Recombinant Salmonella schwarzengrund Elongation factor Ts?

To confirm that recombinant EF-Ts retains its native functionality, researchers should consider implementing the following validation approaches:

• Nucleotide exchange assay: Measure the ability of recombinant EF-Ts to catalyze GDP/GTP exchange on EF-Tu using fluorescently labeled nucleotides or radioactive assays.

• In vitro translation systems: Assess the capacity of recombinant EF-Ts to support protein synthesis in reconstituted translation systems, comparable to approaches used in studies of Salmonella enterica .

• Binding affinity measurements: Determine binding kinetics between recombinant EF-Ts and EF-Tu using techniques such as surface plasmon resonance or isothermal titration calorimetry.

• Structural integrity analysis: Compare secondary structure elements using circular dichroism or thermal shift assays to evaluate proper folding.

• Complementation studies: Test whether the recombinant protein can complement EF-Ts deficiency in controlled systems.

Data from these validation experiments should be represented in tables comparing activity parameters between recombinant and control samples, with statistical analysis of replicate measurements.

How can Recombinant Salmonella schwarzengrund Elongation factor Ts be optimized for structural biology studies?

Structural biology studies require careful optimization of sample preparation and experimental conditions:

Sample Preparation Optimization:

• Purification enhancement: While product information indicates >85% purity by SDS-PAGE , structural studies typically require >95% purity. Additional purification steps such as ion exchange or size exclusion chromatography are recommended.

• Buffer screening: Systematically test different buffer compositions to identify conditions that maximize stability while maintaining native conformation. Starting recommendations include:

  • pH range: 7.0-8.0

  • Salt concentration: 50-200 mM NaCl

  • Stabilizing agents: 5-10% glycerol

• Tag removal considerations: For crystallography studies, consider removing affinity tags using specific proteases if they interfere with crystal formation.

Technique-Specific Considerations:

The reconstitution recommendations provided in the product information (0.1-1.0 mg/mL in deionized sterile water with 5-50% glycerol) serve as a starting point, but optimization for specific structural techniques will be necessary.

What insights can comparative analysis of Elongation factor Ts across different Salmonella serovars provide?

Comparative analysis of Elongation factor Ts across Salmonella serovars can reveal important evolutionary and functional insights:

Sequence Conservation Analysis:

Alignment of EF-Ts sequences from different Salmonella serovars (including S. schwarzengrund, S. Typhimurium, S. Typhi, S. Heidelberg, and S. Minnesota referenced in the search results ) can identify:

• Core conserved regions essential for function

• Variable regions that may contribute to serovar-specific characteristics

• Potential adaptation signatures related to host specificity

Structure-Function Relationships:

This comparative approach aligns with research methodologies seen in Salmonella genomic studies that examine evolutionary relationships between serovars and their virulence characteristics .

How can Recombinant Salmonella schwarzengrund Elongation factor Ts contribute to antimicrobial resistance research?

Recombinant EF-Ts can serve as a valuable tool in antimicrobial resistance research, particularly relevant given the increasing concern about resistance in nontyphoidal Salmonella :

Translation Machinery as Antimicrobial Targets:

• Target-based screening: Develop high-throughput assays using recombinant EF-Ts to identify novel compounds that disrupt its interaction with EF-Tu.

• Resistance mechanism studies: Investigate whether alterations in translation machinery components contribute to resistance phenotypes observed in Salmonella serovars like S. Heidelberg and S. Minnesota, which show high prevalence of resistance genes (sul2, tetA, blaCMY-2) .

• Structure-based drug design: Utilize structural information about EF-Ts to design inhibitors specifically targeting bacterial translation.

Experimental Design Considerations:

These approaches align with broader antimicrobial resistance research in Salmonella, where understanding molecular mechanisms of resistance is crucial for developing new therapeutic strategies .

What are the optimal storage and handling conditions for Recombinant Salmonella schwarzengrund Elongation factor Ts?

Based on product information , researchers should adhere to the following storage and handling protocols:

Storage Recommendations:

• Short-term storage: -20°C

• Extended storage: -80°C

• Working aliquots: 4°C for up to one week

• Shelf life: 6 months for liquid form at -20°C/-80°C; 12 months for lyophilized form

Reconstitution Protocol:

• Centrifuge vial briefly before opening

• Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

• Add glycerol to 5-50% final concentration (50% is recommended) for long-term storage

• Prepare small working aliquots to avoid repeated freeze-thaw cycles

Quality Control Considerations:

Maintaining proper storage conditions is critical for preserving protein functionality, particularly for applications requiring native conformation.

How can researchers design experiments to investigate the role of Elongation factor Ts in Salmonella pathogenesis?

Investigating EF-Ts in Salmonella pathogenesis requires multidisciplinary approaches similar to those used in S. Typhi research :

Genetic Manipulation Strategies:

• Conditional expression systems: Since EF-Ts is likely essential, use inducible promoters to control expression levels.

• Domain-specific mutations: Introduce targeted mutations in functional domains to create variants with altered activity levels.

• Reporter fusions: Create translational fusions to monitor EF-Ts expression and localization during infection.

Infection Model Systems:

Omics Approaches:

• Transcriptomics: Compare gene expression profiles between wild-type and EF-Ts-altered strains using RNA-seq methods similar to those developed for S. Typhi .

• Proteomics: Quantify changes in protein synthesis patterns using mass spectrometry approaches to map peptides back to the genome, as described for S. Typhi .

These experimental approaches would provide comprehensive insights into how EF-Ts contributes to Salmonella pathogenesis beyond its canonical role in translation.

How does Elongation factor Ts from Salmonella schwarzengrund compare with homologs from other bacterial pathogens?

Comparative analysis across bacterial species provides broader evolutionary and functional context:

Cross-Species Homology Analysis:

Functional Conservation Assessment:

• Interchangeability testing: Determine whether EF-Ts from different species can functionally substitute for each other in reconstituted systems.

• Binding partner specificity: Compare interaction profiles with EF-Tu from various species.

• Species-specific inhibition profiles: Assess differential susceptibility to inhibitors or antibiotics.

This comparative approach aligns with systems biology perspectives used in Salmonella research that examine the evolution of virulence mechanisms across the genus .

What methodological considerations are important when using Recombinant Salmonella schwarzengrund Elongation factor Ts in multi-protein complex studies?

Studying EF-Ts in the context of multi-protein complexes requires specific methodological considerations:

Complex Formation and Stability:

• Partner protein co-expression: Consider co-expressing EF-Ts with EF-Tu to improve complex stability.

• Assembly conditions: Optimize buffer conditions, protein ratios, and incubation parameters to promote stable complex formation.

• Interaction mapping: Use hydrogen-deuterium exchange mass spectrometry or crosslinking approaches to identify interaction interfaces.

Analytical Approaches for Complex Characterization:

Functional Reconstitution:

• Translation system assembly: Incorporate purified EF-Ts into reconstituted translation systems with ribosomes, mRNAs, and other factors.

• Dynamic measurements: Use fluorescence-based approaches to monitor complex assembly and disassembly kinetics.

These methodologies are consistent with structural biology approaches used in studying bacterial translation machinery components, as referenced in research on Salmonella enterica proteome characterization .

What are the emerging research directions for understanding the role of Elongation factor Ts in Salmonella virulence and adaptation?

Several promising research directions emerge from current understanding:

• Stress response roles: Investigate whether EF-Ts contributes to adaptation to host environments beyond its canonical translation function.

• Antimicrobial resistance connections: Explore potential links between translation machinery adaptations and the emergence of resistance phenotypes observed in various Salmonella serovars .

• Host-pathogen interface: Examine whether EF-Ts indirectly influences host-pathogen interactions by modulating expression of virulence factors.

• Vaccine development applications: Consider approaches similar to the recombinant S. gallinarum vaccine strategy that could incorporate EF-Ts as an antigen or expression platform.

• Cross-serovar comparative studies: Extend comparative analyses across multiple Salmonella serovars to identify potential correlations between EF-Ts sequence variations and epidemiological characteristics.