Recombinant Klebsiella pneumoniae Elongation factor Ts (tsf)

What is Elongation factor Ts and what is its role in bacterial protein synthesis?

Elongation factor Ts (EF-Ts) serves as a nucleotide exchange factor that catalyzes the regeneration of active EF-Tu by promoting the exchange of GDP for GTP. In K. pneumoniae, the 283-amino acid EF-Ts protein (UniProt ID: A6T4X2) contains specific domains that facilitate interaction with EF-Tu during the translation elongation cycle . This exchange function is critical for maintaining efficient protein synthesis, as it allows EF-Tu to participate in multiple rounds of aminoacyl-tRNA delivery to the ribosome. The protein is encoded by the tsf gene and plays an essential role in bacterial viability through its contribution to translation fidelity and efficiency.

What interactions occur between Elongation factor Ts and Elongation factor Tu during translation?

During bacterial translation, Elongation factor Ts interacts with EF-Tu through a multi-step process:

• EF-Ts recognizes and binds to EF-Tu-GDP after GTP hydrolysis has occurred during aminoacyl-tRNA delivery to the ribosome

• The binding induces conformational changes in EF-Tu that decrease its affinity for GDP

• EF-Ts stabilizes the nucleotide-free form of EF-Tu, facilitating GDP release

• When GTP binds to the complex, structural rearrangements occur that displace EF-Ts

• The regenerated EF-Tu-GTP complex is then ready for another round of aminoacyl-tRNA binding

These protein-protein interactions have been extensively studied in other bacterial systems and appear to be conserved in K. pneumoniae, though species-specific kinetic parameters may vary .

What are the optimal conditions for expressing and purifying recombinant K. pneumoniae Elongation factor Ts?

Based on product information and standard protocols for similar proteins, the following conditions are recommended:

Expression System:

• Host: Escherichia coli (preferably BL21(DE3) or similar strains)

• Vector: T7 promoter-based expression vector with appropriate tag (His-tag commonly used)

• Induction: 0.5-1.0 mM IPTG at OD600 of 0.6-0.8

• Temperature: 18-25°C for 16-20 hours (lower temperatures favor proper folding)

Purification Protocol:

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resin

• Ion exchange chromatography (based on theoretical pI)

• Size exclusion chromatography as a final polishing step

Buffer Considerations:

• Lysis buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 5% glycerol, 1 mM PMSF

• Purification buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol

• Storage: Addition of 50% glycerol for long-term storage at -20°C or -80°C

The purified protein should achieve >85% purity as assessed by SDS-PAGE .

What assays can be used to measure the nucleotide exchange activity of recombinant K. pneumoniae Elongation factor Ts?

Several complementary assays can effectively measure the nucleotide exchange activity:

Fluorescence-Based Assays:

• Mant-GDP displacement assay: Monitors the decrease in fluorescence when fluorescently labeled GDP is displaced from EF-Tu

• FRET-based assays: Using labeled EF-Tu and nucleotides to track conformational changes during exchange

Radioactive Nucleotide Exchange Assays:

• [³H] or [³⁵S]-labeled GDP/GTP exchange measurements

• Filter binding or rapid quench approaches to separate bound from free nucleotides

Real-Time Kinetic Measurements:

• Stopped-flow spectroscopy to measure rapid kinetics of binding and release events

• Surface plasmon resonance or biolayer interferometry to monitor binding kinetics

Data Analysis Considerations:

• Multiple controls should be included (spontaneous exchange rates, temperature effects)

• Kinetic parameters (koff, kon) should be determined under various conditions

• Comparison with EF-Ts from other bacterial species provides valuable context

These methods provide quantitative assessment of the protein's functional activity in vitro.

How can researchers verify that recombinant K. pneumoniae Elongation factor Ts maintains native conformation?

Multiple complementary approaches should be employed to verify native conformation:

Structural Analysis:

• Circular dichroism (CD) spectroscopy to assess secondary structure content

• Thermal shift assays to evaluate protein stability

• Size exclusion chromatography to confirm monomeric state

• Limited proteolysis to identify accessible cleavage sites in properly folded protein

Functional Validation:

• Nucleotide exchange activity with cognate EF-Tu

• Binding affinity determination for EF-Tu using SPR or ITC

• Complex formation analysis using native PAGE or analytical ultracentrifugation

Biophysical Characterization:

• Intrinsic tryptophan fluorescence spectroscopy to monitor tertiary structure

• Dynamic light scattering to assess homogeneity

Properly folded EF-Ts should demonstrate characteristics consistent with its predicted structure and exhibit functional activity comparable to the native protein. These analyses are particularly important when establishing new expression and purification protocols.

How might K. pneumoniae Elongation factor Ts contribute to antibiotic resistance mechanisms?

While Elongation factor Ts itself is not typically associated with direct antibiotic resistance mechanisms, its role in bacterial protein synthesis makes it relevant to antimicrobial resistance in several ways:

• Potential Drug Target: As a critical component of bacterial translation, EF-Ts represents a potential target for novel antibiotics. Inhibiting the EF-Ts:EF-Tu interaction could disrupt protein synthesis and bacterial viability.

• Protein Synthesis Adaptation: In K. pneumoniae strains carrying extended-spectrum β-lactamase (ESBL) genes like those found in the pESBL-PH plasmid , upregulation or modification of translation factors might contribute to adaptive responses under antibiotic stress.

• Evolutionary Considerations: The conservation of EF-Ts across bacterial species, including multidrug-resistant strains, highlights its essential nature and potential as a broad-spectrum target.

• Stress Response: During antibiotic exposure, bacteria often modify their translational machinery. Changes in EF-Ts expression or activity could potentially contribute to stress adaptation mechanisms.

Research exploring these connections would require gene expression studies under antibiotic stress conditions and functional characterization of EF-Ts in resistant versus susceptible strains.

What role might Elongation factor Ts play in K. pneumoniae pathogenesis?

While primarily known for its canonical role in translation, emerging research on bacterial elongation factors suggests potential moonlighting functions relevant to pathogenesis:

• Potential Surface Exposure: Studies have shown that elongation factors in other bacterial pathogens can localize to the cell surface and interact with host components . If K. pneumoniae EF-Ts exhibits similar behavior, it could potentially interact with host molecules.

• Moonlighting Activities: In other bacteria, elongation factors have been found to moonlight as adhesins or immunomodulatory proteins . Investigation of K. pneumoniae EF-Ts for similar functions would require cell binding assays and host-pathogen interaction studies.

• Virulence Regulation: Translation efficiency affects the expression of virulence factors. Changes in EF-Ts activity could potentially modulate virulence gene expression, particularly during infection or stress conditions.

• Biofilm Formation: Protein synthesis regulation plays a role in biofilm development, a key virulence trait of K. pneumoniae. The role of EF-Ts in this process remains to be elucidated.

Research in this area should focus on comparing wild-type and EF-Ts-depleted strains in various infection models and host interaction assays.

How can site-directed mutagenesis be applied to study functional domains of K. pneumoniae Elongation factor Ts?

Site-directed mutagenesis provides a powerful approach for mapping structure-function relationships in K. pneumoniae EF-Ts:

Target Selection Strategy:

• Identify conserved residues through multiple sequence alignment with well-characterized bacterial EF-Ts proteins

• Focus on regions predicted to interact with EF-Tu based on structural modeling

• Target residues in the nucleotide exchange active site

• Select surface-exposed residues potentially involved in protein-protein interactions

Experimental Workflow:

• Generate mutations using PCR-based methods (e.g., QuikChange)

• Express and purify mutant proteins following established protocols

• Assess the impact on:

  • EF-Tu binding using SPR, ITC, or pull-down assays

  • Nucleotide exchange activity using fluorescence-based assays

  • Protein stability using thermal shift assays or CD

  • Structure using X-ray crystallography if possible

Data Analysis Framework:

• Compare kinetic parameters of mutants versus wild-type

• Correlate functional effects with structural information

• Generate comprehensive mutation maps for different functional properties

This approach allows systematic characterization of which residues and regions are critical for specific EF-Ts functions, providing insights for potential therapeutic targeting.

What are common challenges in expressing recombinant K. pneumoniae Elongation factor Ts and how can they be addressed?

Researchers may encounter several challenges when expressing recombinant K. pneumoniae EF-Ts:

Systematic optimization of these parameters typically resolves most expression and purification challenges.

How can researchers interpret discrepancies between in vitro and in vivo studies of K. pneumoniae Elongation factor Ts function?

When discrepancies arise between in vitro biochemical studies and in vivo observations of EF-Ts function, consider the following interpretive framework:

Sources of Discrepancies:

• Environmental Differences:

  • Macromolecular crowding effects in the cellular environment

  • Differences in ionic conditions and pH between test tube and cytoplasm

  • Presence of additional binding partners in vivo

• Methodological Considerations:

  • Recombinant protein may lack post-translational modifications present in vivo

  • In vitro conditions typically use simplified component systems

  • Time scales of measurements often differ between approaches

Reconciliation Strategies:

• Bridge the Gap:

  • Develop more complex in vitro systems (reconstituted translation systems)

  • Use cellular extracts for semi-in vivo approaches

  • Perform in-cell measurements when possible

• Validation Approaches:

  • Create conditional mutant strains to correlate phenotypes with biochemical activities

  • Compare wild-type versus mutant complementation

  • Design targeted in vivo assays that monitor specific EF-Ts functions

• Interpretive Framework:

  • Identify whether discrepancies are quantitative (magnitude) or qualitative (mechanism)

  • Consider whether additional factors might regulate EF-Ts in vivo

  • Determine if observed differences reveal novel biological insights

Discrepancies often provide valuable clues about regulatory mechanisms and contextual factors affecting protein function in the cellular environment.

How might structural studies of K. pneumoniae Elongation factor Ts contribute to new antimicrobial development?

Structural studies of K. pneumoniae EF-Ts could significantly advance antimicrobial development through several avenues:

• Structure-Based Drug Design:

  • High-resolution structures could reveal unique binding pockets suitable for small molecule inhibitors

  • Comparative analysis with human elongation factors would help identify bacterial-specific features

  • Molecular dynamics simulations could identify transient pockets for allosteric inhibitors

• Targeting Protein-Protein Interactions:

  • Mapping the EF-Ts:EF-Tu interface could enable development of peptide mimetics or small molecules that disrupt this essential interaction

  • Identification of hot spots within the interface would prioritize specific regions for targeting

• Novel Screening Approaches:

  • Structural information enables fragment-based drug discovery approaches

  • Virtual screening against identified pockets could rapidly identify lead compounds

  • Structure-guided design of biosensors for high-throughput screening

• Resistance Considerations:

  • Structural comparison across multiple clinical isolates could identify conserved features less prone to resistance-conferring mutations

  • Understanding the structural basis of EF-Ts function could help predict and counter potential resistance mechanisms

Given the rising prevalence of multidrug-resistant K. pneumoniae, including ESBL-producing strains , developing novel antimicrobials targeting essential factors like EF-Ts represents a promising alternative to traditional antibiotic classes.

What potential exists for using K. pneumoniae Elongation factor Ts in synthetic biology applications?

K. pneumoniae Elongation factor Ts offers several intriguing possibilities for synthetic biology applications:

• Enhanced Protein Production Systems:

  • Co-expression of optimized EF-Ts could potentially increase translation efficiency in heterologous expression systems

  • Engineering EF-Ts variants with enhanced nucleotide exchange activity might improve protein synthesis rates

• Biosensors and Diagnostic Tools:

  • EF-Ts:EF-Tu interactions could be leveraged to design FRET-based biosensors for nucleotide exchange activity

  • Species-specific variants could potentially be used in diagnostic applications for K. pneumoniae detection

• Minimal Cell Systems:

  • As an essential component of the translation machinery, optimized EF-Ts variants could be incorporated into minimal cell designs

  • Understanding the minimal functional requirements of EF-Ts could inform the design of simplified translation systems

• Protein Interaction Scaffolds:

  • The structured nature of EF-Ts and its ability to form stable complexes with EF-Tu could be exploited as a scaffold for organizing other protein components in synthetic systems

• Directed Evolution Platforms:

  • EF-Ts could serve as a target for directed evolution approaches aimed at understanding protein-protein interaction evolution

  • Selection systems based on EF-Ts function could identify novel regulatory mechanisms

These applications would require detailed structural and functional characterization of K. pneumoniae EF-Ts, including identification of modulatory regions that could be engineered for specific purposes.