Recombinant Escherichia coli O9:H4 Elongation factor Ts (tsf)

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

Elongation factor Ts (EF-Ts) is a guanine nucleotide exchange factor that plays a critical role in protein synthesis by facilitating the regeneration of active EF-Tu·GTP from inactive EF-Tu·GDP. Recent research has revealed that EF-Ts not only catalyzes nucleotide exchange but also directly facilitates both the formation and dissociation of the EF-Tu·GTP·aminoacyl-tRNA ternary complex essential for translation elongation . This dual functionality makes EF-Ts central to maintaining efficient translation rates in bacteria. The protein works by binding to EF-Tu·GDP, causing conformational changes that lead to GDP release, followed by GTP binding and subsequent release of EF-Ts, allowing EF-Tu·GTP to participate in another round of aminoacyl-tRNA delivery to the ribosome .

Where is the tsf gene located in the E. coli genome?

The structural gene for elongation factor EF-Ts (tsf) has been mapped near dapD at approximately 4 minutes on the E. coli genetic map . This location is notably different from the chromosomal locations where many other translation-related genes cluster, such as the str-spc region and the rif region, which contain numerous ribosomal protein genes, RNA polymerase components, and other elongation factor genes (fus, tufA, and tufB) . The tsf gene is co-located with the rpsB gene, which encodes the ribosomal protein S2, suggesting possible co-regulation of these translation factors . Both genes have been identified through lambda transducing phages that were isolated as dapD+polC+ transducing phages, allowing for the precise mapping and identification of these essential genes .

How does EF-Ts structurally interact with EF-Tu?

EF-Ts interacts with EF-Tu through specific domain contacts that have been identified through mutation studies and structural analyses. Research has identified nine single-amino acid substitution mutations in the region 146-199 of EF-Tu that affect its interaction with EF-Ts . These mutations (R154C, P168L, A174V, K176E, D181G, E190K, D196G, S197F, and I199V) were discovered through a suppression screening method based on the EF-Tu dominant negative mutation K136E, which exerts its effect by sequestering EF-Ts . The structural interface between these proteins involves complementary surfaces that facilitate both binding and subsequent release, essential for the cycling of EF-Tu between its GDP- and GTP-bound states during protein synthesis. The identified mutations affect this interaction by altering the binding affinity or conformational dynamics of the complex.

What are the optimal storage conditions for recombinant E. coli EF-Ts?

Recombinant E. coli Elongation factor Ts should be stored at -20°C for regular use, while extended storage requires conservation at -20°C or -80°C to maintain protein stability and activity . The protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL with the addition of 5-50% glycerol (final concentration) as a cryoprotectant before aliquoting for long-term storage . Repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and loss of activity. The shelf life of liquid preparations is approximately 6 months at -20°C/-80°C, while lyophilized forms can maintain stability for about 12 months at the same temperatures . For short-term use, working aliquots can be stored at 4°C for up to one week without significant loss of activity .

How can I express and purify recombinant EF-Ts with high yield and purity?

The expression and purification of recombinant EF-Ts from E. coli require strategic approaches to ensure high yield and purity. A recommended protocol involves:

• Expression System Selection: Use a baculovirus expression system, which has proven effective for producing full-length mature EF-Ts protein (amino acids 2-283) .

• Vector Construction: Design an expression vector with appropriate promoter elements and purification tags, ensuring the tag type is compatible with the protein's structure and function.

• Expression Conditions: Optimize temperature, IPTG concentration, and induction time to maximize protein expression while minimizing inclusion body formation.

• Cell Lysis: Use gentle lysis methods (e.g., sonication with pulse cycles) in buffer systems containing protease inhibitors to prevent degradation.

• Purification Strategy: Implement a multi-step purification process:

  • Initial capture using affinity chromatography based on the chosen tag

  • Intermediate purification using ion-exchange chromatography

  • Polishing step with size-exclusion chromatography to achieve >85% purity as assessed by SDS-PAGE

• Quality Assessment: Verify protein identity and purity using mass spectrometry and SDS-PAGE, respectively.

This approach yields EF-Ts with the full sequence (AEITASLVKELRERTGAGMMDCKKALTEA through AEVAAMSKQS) as confirmed by sequence analysis .

What methods are most effective for studying EF-Ts interactions with EF-Tu in vitro?

Several complementary methodologies have proven effective for studying the interactions between EF-Ts and EF-Tu in vitro:

• Suppression Genetics: This approach identifies interaction domains by screening for suppressor mutations that restore function. For example, researchers have successfully used EF-Tu dominant negative mutation K136E (which sequesters EF-Ts) to identify nine single amino acid substitutions in the 146-199 region of EF-Tu that affect interaction with EF-Ts .

• Fluorescence-Based Assays: These techniques utilize fluorescently labeled nucleotides or proteins to monitor:

  • Nucleotide exchange kinetics in real-time

  • Association and dissociation rates between EF-Tu and EF-Ts

  • Conformational changes during complex formation

• Surface Plasmon Resonance (SPR): Provides quantitative binding data including association/dissociation rate constants and equilibrium dissociation constants.

• Isothermal Titration Calorimetry (ITC): Measures thermodynamic parameters of binding interactions.

• Cryo-Electron Microscopy or X-ray Crystallography: Offers structural insights into the EF-Tu:EF-Ts complex at near-atomic resolution.

• Biochemical Assays: Measures functional outcomes such as:

  • GDP/GTP exchange rates

  • Ternary complex formation rates with aminoacyl-tRNAs

  • Effects on translation efficiency in reconstituted translation systems

Recent studies have particularly highlighted the value of combining these approaches to reveal that EF-Ts not only facilitates nucleotide exchange but also directly affects ternary complex formation and dissociation .

How does EF-Ts respond to stress conditions, and what are the implications for bacterial adaptation?

Under stress conditions, particularly protein denaturation stress, E. coli shows a remarkable adaptation involving EF-Ts. Two-dimensional electrophoresis studies have revealed that when E. coli is exposed to the protein denaturant guanidine hydrochloride, the expression level of EF-Ts increases approximately 1.61-fold compared to non-stress conditions . This upregulation occurs against a backdrop of widespread protein aggregation and decreased synthesis, where 34 out of 699 soluble proteins disappear entirely and 63 proteins decrease by over 2.5-fold .

The stress-induced increase in EF-Ts suggests its important role in maintaining translational capacity under adverse conditions. This adaptive response may allow bacteria to:

• Sustain protein synthesis despite the accumulation of damaged proteins

• Maintain critical cellular functions by ensuring efficient elongation during translation

• Potentially aid in proper folding of newly synthesized proteins

These findings point to EF-Ts having a previously unrecognized role in stress response pathways, positioning it as not merely a translation factor but also a stress-responsive protein that helps maintain cellular homeostasis under challenging environmental conditions .

Can EF-Ts be used as a fusion partner to enhance recombinant protein solubility, and what are the mechanistic details?

EF-Ts has emerged as an effective fusion partner for enhancing the solubility of heterologous proteins in E. coli. Research has demonstrated that N-terminal fusions of EF-Ts can dramatically improve the soluble expression of various recombinant proteins that would otherwise form inclusion bodies . The mechanism appears to involve EF-Ts's intrinsically high folding efficiency, which allows it to sequester interactive surfaces of heterologous proteins from engaging in nonspecific protein-protein interactions that lead to aggregation .

The effectiveness of EF-Ts as a solubility enhancer has been validated with multiple test proteins, including a biologically active bacterial cutinase that retained its enzymatic activity in the fusion construct . This application holds significant potential for biotechnology and commercial applications where protein solubility is often a limiting factor.

Mechanistically, the EF-Ts fusion approach works through:

• Providing a well-folded domain that initiates proper folding of the target protein

• Shielding hydrophobic patches on the target protein that might otherwise drive aggregation

• Potentially enhancing the interaction with cellular chaperones to promote correct folding

• Possibly slowing the translation rate of the fusion construct, allowing more time for proper folding

This approach represents an important addition to the toolkit for recombinant protein expression, particularly for challenging targets that resist conventional solubilization strategies .

What is the precise mechanism by which EF-Ts facilitates ternary complex formation and dissociation?

Recent research has uncovered a novel function of EF-Ts beyond its established role as a guanine nucleotide exchange factor. Studies have shown that EF-Ts directly facilitates both the formation and dissociation of the ternary complex (EF-Tu·GTP·aminoacyl-tRNA) . This discovery expands our understanding of translation regulation at the elongation phase.

The mechanism involves several coordinated steps:

• Ternary Complex Formation Acceleration: EF-Ts increases the rate at which EF-Tu·GTP binds to aminoacyl-tRNA, effectively acting as a catalyst for ternary complex assembly .

• Conformational Optimization: EF-Ts likely induces subtle conformational changes in EF-Tu that optimize its interaction surface for aminoacyl-tRNA binding.

• Dynamic Cycling Enhancement: By also accelerating ternary complex dissociation, EF-Ts ensures efficient cycling of translation factors, preventing bottlenecks in the elongation phase .

• Integrated Regulation: This dual functionality allows EF-Ts to fine-tune translation rates in response to cellular conditions, potentially serving as an additional regulatory node in protein synthesis.

This mechanism represents a significant paradigm shift in our understanding of translation factor dynamics, suggesting that EF-Ts serves as a multifunctional regulator rather than simply a nucleotide exchange factor . The findings highlight the sophisticated regulatory networks governing bacterial protein synthesis and offer new possibilities for antibiotic development targeting these previously unrecognized interactions.

What are the key considerations when designing experiments to study EF-Ts function in translation systems?

When designing experiments to study EF-Ts function in translation systems, researchers should consider several critical factors:

• Component Purity and Activity:

  • Use highly purified components (>85% purity by SDS-PAGE) for recombinant EF-Ts

  • Verify activity of all translation components before experiments

  • Consider using tagged versions of components for tracking, while ensuring tags don't interfere with function

• Physiological Relevance:

  • Maintain appropriate molar ratios between EF-Ts, EF-Tu, and other components (typically, EF-Tu is in excess over EF-Ts in cells)

  • Use buffer conditions that mimic cellular environment (pH, ionic strength, Mg²⁺ concentration)

  • Include molecular crowding agents when appropriate to simulate cytoplasmic conditions

• Kinetic Considerations:

  • Design time-course experiments to capture the rapid kinetics of EF-Ts-mediated reactions

  • Use pre-steady-state kinetics approaches to resolve individual steps in nucleotide exchange

  • Consider temperature effects, as EF-Ts activity is temperature-dependent

• Control Experiments:

  • Include EF-Ts mutants with altered activity as controls

  • Compare results with and without EF-Ts to quantify its contribution

  • Validate in vitro findings with complementary in vivo approaches

• System Complexity:

  • Begin with simplified systems examining only EF-Ts and EF-Tu interaction

  • Gradually increase complexity by adding aminoacyl-tRNAs, ribosomes, and other factors

  • Consider reconstituted translation systems for comprehensive functional studies

These considerations ensure robust, physiologically relevant data that accurately captures the multifaceted roles of EF-Ts in translation .

How can researchers differentiate between the nucleotide exchange and ternary complex effects of EF-Ts?

Differentiating between EF-Ts's roles in nucleotide exchange and ternary complex dynamics requires specialized experimental approaches:

• Temporal Separation Experiments:

  • Use rapid kinetics techniques (stopped-flow fluorescence, quench-flow) to resolve fast reactions

  • Design experiments with sequential addition of components to isolate individual steps

  • Employ time-resolved measurements to distinguish primary from secondary effects

• Component Variation Strategies:

  • Perform experiments with variable concentrations of GTP/GDP to isolate nucleotide exchange effects

  • Vary aminoacyl-tRNA concentrations while maintaining constant nucleotide levels to focus on ternary complex effects

  • Create a nucleotide exchange-deficient EF-Ts mutant to isolate its direct effects on ternary complex

• Specialized Assays:

  • Use fluorescent nucleotide analogs (mant-GDP/GTP) to directly monitor nucleotide exchange

  • Employ fluorescently labeled aminoacyl-tRNAs to track ternary complex formation independently

  • Implement FRET-based assays between labeled EF-Tu and aminoacyl-tRNA to monitor their interaction specifically

• Mathematical Modeling:

  • Develop kinetic models that incorporate both functions

  • Fit experimental data to these models to extract rate constants for individual steps

  • Use sensitivity analysis to determine which function predominates under different conditions

These approaches allow researchers to deconvolute the dual functions of EF-Ts and quantify their relative contributions to translation efficiency under various conditions .

What are the most common pitfalls when working with recombinant EF-Ts, and how can they be avoided?

Working with recombinant Elongation factor Ts presents several challenges that researchers should be aware of:

• Protein Stability Issues:

  • Pitfall: Activity loss during storage due to protein denaturation

  • Solution: Store at -20°C or -80°C with 5-50% glycerol as a cryoprotectant; avoid repeated freeze-thaw cycles; prepare working aliquots for short-term use at 4°C (up to one week)

• Purification Challenges:

  • Pitfall: Co-purification of contaminating E. coli proteins with similar properties

  • Solution: Implement multi-step purification strategies; verify purity by SDS-PAGE (>85% purity should be the target); confirm protein identity by mass spectrometry or N-terminal sequencing

• Activity Verification:

  • Pitfall: Using structurally intact but functionally compromised protein

  • Solution: Perform activity assays measuring nucleotide exchange rates or ternary complex formation before experimental use

• Buffer Compatibility:

  • Pitfall: Protein aggregation or activity loss in experimental buffers

  • Solution: Perform buffer optimization; monitor protein stability using dynamic light scattering or thermal shift assays; consider adding stabilizing agents (glycerol, reducing agents)

• Tag Interference:

  • Pitfall: Purification tags affecting EF-Ts function or interactions

  • Solution: Compare tagged and untagged versions in functional assays; position tags to minimize interference; consider tag removal if necessary

• Concentration Determination:

  • Pitfall: Inaccurate protein quantification leading to inconsistent results

  • Solution: Use multiple quantification methods (Bradford, BCA, absorbance at 280nm with known extinction coefficient); validate with SDS-PAGE and densitometry

• Reconstitution Errors:

  • Pitfall: Improper reconstitution leading to inactive protein

  • Solution: Follow established protocols for reconstituting protein in deionized sterile water to concentrations of 0.1-1.0 mg/mL

By anticipating these challenges, researchers can maximize the quality and reliability of their experiments with recombinant EF-Ts.

How does EF-Ts structure and function compare between different bacterial species?

Elongation factor Ts exhibits notable structural and functional conservation across bacterial species, with important variations that reflect evolutionary adaptation:

These comparisons provide insights into both the essential core functions that must be maintained across all bacteria and the specific adaptations that contribute to bacterial survival in diverse environments.

What insights can comparative genomics provide about the evolution and conservation of the tsf gene?

Comparative genomics analyses of the tsf gene, encoding Elongation factor Ts, reveal important insights into its evolution and conservation:

• Genomic Context:

  • The tsf gene in E. coli is located near dapD at approximately 4 minutes on the genetic map

  • Unlike many translation-related genes that cluster in the str-spc region or rif region, tsf has a distinct genomic context

  • The genomic neighborhood of tsf often includes rpsB (encoding ribosomal protein S2), suggesting potential co-regulation or functional relationships

  • This genomic arrangement is widely conserved across diverse bacterial lineages, indicating an ancient gene organization

• Sequence Conservation:

  • Core functional domains show high sequence conservation across bacterial phyla

  • The EF-Tu binding interface exhibits the highest level of conservation

  • N-terminal and C-terminal regions show greater sequence divergence

  • Specific protein regions have evolved at different rates, reflecting varying selective pressures

• Evolutionary Patterns:

  • tsf belongs to a set of highly conserved genes considered part of the minimal bacterial genome

  • Horizontal gene transfer events involving tsf appear rare, consistent with its central role in translation

  • Evolutionary rate analysis suggests purifying selection as the dominant force shaping tsf evolution

  • Species-specific adaptations are more prevalent in regions not directly involved in EF-Tu interaction

• Structural Conservation:

  • Secondary structure elements are more conserved than primary sequence

  • Critical interaction surfaces maintain spatial conservation despite sequence variations

  • Thermophilic adaptations often involve increased hydrophobic core packing and surface salt bridges

These genomic insights provide a framework for understanding the evolutionary constraints on this essential translation factor and help identify regions that may be targeted for antimicrobial development with minimal risk of resistance development.

What are the emerging applications of EF-Ts in biotechnology beyond its role in translation?

Elongation factor Ts is emerging as a versatile tool in biotechnology with applications extending beyond its traditional role in translation:

• Protein Solubility Enhancement:

  • EF-Ts has demonstrated effectiveness as a fusion partner for improving the solubility of recombinant proteins in E. coli

  • This approach has successfully produced soluble versions of proteins that typically form inclusion bodies when expressed directly

  • The application has been validated with various heterologous proteins, including an active bacterial cutinase

  • The intrinsic high folding efficiency of EF-Ts enables it to shield interactive surfaces of target proteins from nonspecific interactions

• Protein Engineering Platform:

  • The structural stability of EF-Ts makes it an attractive scaffold for protein engineering

  • Its natural ability to interact with other proteins can be exploited for creating novel binding interfaces

  • The well-characterized structure allows for rational design of modified versions with enhanced functions

• Cell-Free Protein Synthesis Enhancement:

  • Addition of optimized concentrations of EF-Ts to cell-free protein synthesis systems can improve yield and efficiency

  • Engineered variants of EF-Ts with enhanced stability or altered specificity can further improve these systems

  • This application is particularly valuable for the production of difficult-to-express proteins

• Stress Response Studies:

  • The upregulation of EF-Ts under stress conditions (1.61-fold increase under guanidine hydrochloride stress) suggests its potential use as a stress biomarker

  • This property could be harnessed for developing cellular stress sensors or reporters

  • Understanding the stress-responsive regulation of EF-Ts may provide insights into bacterial adaptation mechanisms

These emerging applications highlight the versatility of EF-Ts beyond its canonical role and suggest exciting new directions for biotechnology research and development .

How might understanding EF-Ts function contribute to new antimicrobial development strategies?

Understanding the structure and function of Elongation factor Ts offers several promising avenues for novel antimicrobial development:

• Direct Inhibition Strategies:

  • Targeting the EF-Ts:EF-Tu interface with small molecules could disrupt nucleotide exchange, stalling bacterial protein synthesis

  • Compounds that lock EF-Ts in complex with EF-Tu would prevent the recycling of translation factors

  • Structure-based drug design using the known interaction domains between EF-Ts and EF-Tu can guide rational inhibitor development

• Pathway-Based Approaches:

  • The newly discovered role of EF-Ts in ternary complex formation/dissociation presents additional targeting opportunities beyond nucleotide exchange

  • Molecules that selectively alter one function while preserving others could create translation bottlenecks

  • Targeting the regulatory mechanisms controlling EF-Ts expression, particularly under stress conditions, might sensitize bacteria to environmental challenges

• Species-Specific Targeting:

  • Structural differences in EF-Ts between bacterial species can be exploited for narrow-spectrum antibiotics

  • Similar to the approach used with O-antigen tailspike-R-type pyocins against E. coli O104:H4, species-specific elements of EF-Ts could be targeted

  • This precision approach could minimize disruption of beneficial microbiota

• Resistance Considerations:

  • The essential nature and high conservation of EF-Ts suggest a high genetic barrier to resistance

  • Mutations affecting drug binding would likely compromise EF-Ts function

  • Combination approaches targeting multiple aspects of translation might further reduce resistance development

These strategies represent promising directions for addressing the growing challenge of antimicrobial resistance by targeting the fundamental process of bacterial protein synthesis at a previously underexploited node .

Table 1: Properties of Recombinant E. coli Elongation Factor Ts

Source: Data compiled from product specifications and research literature .

Table 2: Functional Activities of EF-Ts in Different Contexts