Recombinant Bacillus licheniformis Elongation factor 4 (lepA), partial

What is Elongation Factor 4 (lepA) and what is its function in Bacillus licheniformis?

Elongation Factor 4, also known as LepA or EF-4, is a highly conserved bacterial protein that functions as a ribosomal back-translocase during protein synthesis. In Bacillus licheniformis, as in other bacteria, lepA plays a critical role in ensuring translation fidelity by catalyzing a reverse translocation reaction when errors occur during protein synthesis. This protein belongs to the GTPase superfamily and has an EC classification of 3.6.5.n1, indicating its role in energy-coupled translocation processes . The protein's ability to move ribosomes backward along mRNA allows for error correction mechanisms that improve the accuracy of protein synthesis, particularly under stress conditions.

What expression systems are recommended for recombinant Bacillus licheniformis lepA production?

Multiple expression systems can be utilized for the production of recombinant Elongation Factor 4 from Bacillus licheniformis, each with distinct advantages. E. coli and yeast expression systems typically offer the highest yields and shorter turnaround times, making them cost-effective choices for initial studies . For applications requiring post-translational modifications necessary for proper protein folding or activity retention, insect cells with baculovirus or mammalian cell expression systems are recommended . When selecting an expression system, researchers should consider the specific requirements of their downstream applications, particularly whether native protein conformation is critical to the planned experiments.

How should recombinant Bacillus licheniformis lepA be stored to maintain stability?

The shelf life and stability of recombinant lepA depend on several factors including storage buffer composition, temperature, and the intrinsic stability of the protein itself. For optimal preservation, liquid formulations should be stored at -20°C or -80°C, where they typically maintain stability for approximately 6 months . Lyophilized (freeze-dried) formulations offer extended stability, with a shelf life of up to 12 months when stored at -20°C or -80°C . To minimize protein degradation, repeated freeze-thaw cycles should be avoided. For working solutions, storage at 4°C is acceptable for up to one week . When reconstituting lyophilized protein, it is advisable to use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL, with the addition of 5-50% glycerol (final concentration) for aliquots intended for long-term storage at -20°C/-80°C .

What is the relationship between Bacillus licheniformis as a probiotic and its specific proteins?

Bacillus licheniformis is recognized as a probiotic organism with beneficial effects on both humans and animals . While the specific contribution of Elongation Factor 4 to these probiotic properties is not directly established in the search results, the bacterium itself has been demonstrated to improve growth performance, intestinal mucosal barrier functions, and immunity in animal models . In studies with weaned piglets challenged with lipopolysaccharide (LPS), B. licheniformis supplementation significantly reduced inflammatory responses, enhanced antioxidant activities, and improved immunoglobulin levels . The bacterium's ability to modulate the immune system is evident through its capacity to increase anti-inflammatory factors (IL-10) while decreasing pro-inflammatory factors (TNF-α, IL-6, IL-1β) . These properties make B. licheniformis a valuable subject for research in immune modulation and gut health.

How does post-translational modification affect the activity of recombinant B. licheniformis lepA?

Post-translational modifications (PTMs) play a crucial role in determining the functional activity of recombinant Elongation Factor 4 from Bacillus licheniformis. When expressed in prokaryotic systems like E. coli, the protein lacks eukaryotic-specific modifications that may be essential for optimal folding or activity in certain experimental contexts . For applications requiring native-like activity, expression in insect cells with baculovirus or mammalian cells is recommended as these systems can provide many of the post-translational modifications necessary for correct protein folding and activity retention . The specific PTMs that affect lepA activity have not been fully characterized in the available search results, but may include phosphorylation, methylation, or other modifications that influence protein conformation and GTPase activity. Researchers investigating structure-function relationships should carefully consider the expression system chosen based on the required authenticity of the recombinant protein.

What methodologies are most effective for studying the interaction between B. licheniformis lepA and the ribosome?

Investigating the interaction between Bacillus licheniformis Elongation Factor 4 and the ribosome requires sophisticated biochemical and biophysical approaches. Ribosome binding assays using purified recombinant lepA (>85% purity by SDS-PAGE) and isolated ribosomes can provide initial insights into binding affinities and kinetics. Cryo-electron microscopy has emerged as a powerful technique for visualizing the lepA-ribosome complex at near-atomic resolution, revealing conformational changes that occur during back-translocation. For functional studies, GTP hydrolysis assays can measure the enzymatic activity of lepA during interaction with ribosomes. Additionally, site-directed mutagenesis of specific lepA domains combined with these assays can identify critical residues involved in ribosome binding and catalytic activity. Cross-linking studies followed by mass spectrometry analysis can map precise interaction sites between lepA and ribosomal components. Researchers should design experimental protocols that incorporate appropriate controls and consider the effect of buffer conditions, especially magnesium concentration, which significantly affects ribosome stability and lepA binding.

What is the role of B. licheniformis lepA in stress response and antibiotic resistance?

Elongation Factor 4 in bacteria, including Bacillus licheniformis, has been implicated in cellular responses to various stress conditions. Under stress, lepA's back-translocation activity may become particularly important for maintaining translation accuracy. While not directly addressed in the provided search results, research with related bacterial species suggests that lepA may play a role in adaptation to thermal stress, oxidative stress, and nutrient limitation. The protein's involvement in antibiotic resistance mechanisms is an emerging area of research, particularly regarding antibiotics that target the translation machinery. The expression of lepA may be upregulated under antibiotic exposure, potentially contributing to translation fidelity under these challenging conditions. Understanding this relationship could have significant implications for addressing antimicrobial resistance. Researchers investigating this aspect should design experiments that measure lepA expression levels under various stress conditions using quantitative PCR and evaluate the phenotypic consequences of lepA knockout or overexpression on antibiotic susceptibility profiles.

How can structural information about B. licheniformis lepA inform protein engineering efforts?

Structural characterization of Bacillus licheniformis Elongation Factor 4 provides valuable insights for protein engineering initiatives. While specific structural data for B. licheniformis lepA is not provided in the search results, the protein is likely to share the conserved domains found in lepA proteins from other bacterial species: a GTP-binding domain, a middle domain involved in ribosome interaction, and a C-terminal domain with unique features not found in other translation factors. Advanced structural techniques such as X-ray crystallography, NMR spectroscopy, or cryo-EM can elucidate the precise three-dimensional arrangement of these domains. This structural information can guide rational design approaches for enhancing stability, modifying substrate specificity, or introducing novel functions. Protein engineers might focus on optimizing the GTPase activity through targeted mutations in the GTP-binding pocket or altering ribosome binding specificity by modifying the interface residues. Chimeric proteins incorporating domains from lepA and other translation factors could potentially exhibit novel activities useful for biotechnological applications or as research tools for studying translation mechanisms.

What are the optimal conditions for expression and purification of active recombinant B. licheniformis lepA?

The production of active recombinant Bacillus licheniformis Elongation Factor 4 requires careful optimization of expression and purification conditions. For E. coli-based expression systems, which offer high yields and rapid production cycles , induction parameters (IPTG concentration, temperature, and duration) should be optimized to balance protein yield with proper folding. Lower induction temperatures (16-25°C) often favor correct folding over maximum expression. For purification, a multi-step approach typically yields the best results:

• Initial clarification of cell lysate by centrifugation (12,000×g for 10 minutes)

• Affinity chromatography using an appropriate tag (the specific tag type for lepA should be determined during the manufacturing process)

• Size exclusion chromatography to remove aggregates and improve purity beyond the standard >85% achieved by SDS-PAGE

• Ion exchange chromatography for removing host cell proteins with different charge properties
  Buffer optimization is critical throughout the purification process. For lepA stability, buffers typically contain 20-50 mM Tris-HCl (pH 7.5-8.0), 100-300 mM NaCl, 5-10% glycerol, and 1-5 mM DTT or β-mercaptoethanol. Addition of 1-5 mM MgCl₂ is often beneficial for maintaining the native conformation of GTPase proteins like lepA.

How can the activity of purified recombinant B. licheniformis lepA be accurately measured in vitro?

Assessing the activity of purified recombinant Bacillus licheniformis Elongation Factor 4 requires specialized assays that evaluate its back-translocation function and GTPase activity. A comprehensive approach would include:

• GTPase Activity Assay: Measuring the hydrolysis of GTP by lepA using colorimetric methods (malachite green assay) or radioactive [γ-32P]GTP. The typical reaction conditions include 1-5 μg purified lepA, 50-200 μM GTP, 20 mM Tris-HCl (pH 7.5), 100 mM KCl, and 5-10 mM MgCl₂.

• Ribosome-Dependent GTPase Assay: Since lepA's GTPase activity is stimulated by ribosomes, measuring activity in the presence of purified ribosomes provides insight into functional interactions.

• Back-Translocation Assay: Utilizing dual-labeled ribosome complexes (with fluorescent reporters on tRNA and mRNA) to directly observe lepA-mediated reverse movement using fluorescence resonance energy transfer (FRET).

• Binding Assays: Surface plasmon resonance (SPR) or microscale thermophoresis (MST) to determine binding affinities between lepA and ribosomes or GTP.
  Quality control parameters should include verification of protein concentration using the Bradford or BCA assay, assessment of purity by SDS-PAGE, and confirmation of proper folding through circular dichroism spectroscopy.

What approaches can be used to investigate the structure-function relationship of B. licheniformis lepA?

Investigating the structure-function relationship of Bacillus licheniformis Elongation Factor 4 requires an integrated approach combining genetic, biochemical, and biophysical techniques:

• Expression level and solubility

• Purification yield and purity (>85% by SDS-PAGE as standard)

• Structural integrity via circular dichroism

• GTPase activity with and without ribosomes

• Ribosome binding affinity

• Back-translocation activity
  This comprehensive analysis will reveal key residues and structural elements essential for lepA function.

What are the considerations for designing in vivo studies to assess B. licheniformis lepA function?

Designing in vivo studies to investigate Bacillus licheniformis Elongation Factor 4 function requires careful consideration of several experimental parameters. Based on approaches used in related probiotic research , the following framework is recommended:

• Model System Selection:

  • Bacterial models: B. licheniformis genetic manipulation systems for gene deletion, complementation, or overexpression

  • Animal models: Systems like the weaned piglet model used for B. licheniformis studies , which allows assessment of physiological impacts

• Knockout and Complementation Strategy:

  • Generation of lepA deletion strains using CRISPR-Cas9 or homologous recombination

  • Complementation with wild-type or mutant lepA to confirm phenotypes

  • Controlled expression using inducible promoters

• Stress Challenge Models:

  • Temperature stress (37.8°C vs. elevated temperatures)

  • Oxidative stress (H₂O₂ exposure)

  • Antibiotic challenge (sublethal concentrations)

  • Nutrient limitation

  • LPS challenge (100 μg/kg) as used in B. licheniformis studies

• Phenotypic Assessment:

  • Growth rate determination under various conditions

  • Protein synthesis rate and accuracy measurement

  • Stress response indicator analysis:

    • Antioxidant enzyme activities (GSH-Px, SOD, T-AOC)

    • Inflammatory marker expression (TNF-α, IL-6, IL-1β, IL-10)

    • NLRP3 inflammasome activation

• Controls and Validation:

  • Include multiple control groups (e.g., non-challenged wild type, challenged wild type)

  • Verification of lepA expression levels using qRT-PCR and Western blot

  • Statistical design with sufficient biological replicates (n≥12 based on similar studies)
    This comprehensive approach allows for rigorous assessment of lepA's physiological role in B. licheniformis under various conditions.

What analytical methods are most appropriate for characterizing recombinant B. licheniformis lepA?

Comprehensive characterization of recombinant Bacillus licheniformis Elongation Factor 4 requires multiple analytical approaches to assess its physical, chemical, and functional properties:

• Purity Assessment:

  • SDS-PAGE with Coomassie or silver staining (standard is >85% purity)

  • Size exclusion HPLC or FPLC

  • Capillary electrophoresis

• Identity Confirmation:

  • Western blotting with specific antibodies

  • Peptide mass fingerprinting using tryptic digestion followed by mass spectrometry

  • N-terminal sequencing

• Physical Characterization:

  • Dynamic light scattering (DLS) for hydrodynamic radius and aggregation assessment

  • Analytical ultracentrifugation for oligomeric state determination

  • Circular dichroism spectroscopy for secondary structure analysis

  • Thermal shift assays for stability assessment

• Functional Analysis:

  • GTPase activity assays with colorimetric detection

  • Ribosome binding assays using surface plasmon resonance or microscale thermophoresis

• Post-translational Modification Analysis:

  • Mass spectrometry for identifying and mapping modifications

  • Phosphoprotein-specific staining for phosphorylation detection

  • Glycoprotein staining to detect glycosylation
    Each analytical method should be validated with appropriate controls and standards. Data from multiple orthogonal techniques should be integrated to build a comprehensive profile of the recombinant protein's characteristics.

How do you troubleshoot expression and activity issues with recombinant B. licheniformis lepA?

Troubleshooting expression and activity issues with recombinant Bacillus licheniformis Elongation Factor 4 requires a systematic approach:
For Expression Issues:

• Low Yield

  • Optimize codon usage for the expression host

  • Test different promoter strengths

  • Adjust induction parameters (temperature, inducer concentration, duration)

  • Try different E. coli strains or consider alternative hosts like yeast

• Insoluble Protein/Inclusion Bodies

  • Lower expression temperature (16-25°C)

  • Reduce inducer concentration

  • Co-express with molecular chaperones

  • Try expression in insect or mammalian cells for better folding

• Degradation

  • Add protease inhibitors during lysis

  • Use protease-deficient host strains

  • Optimize buffer conditions
    For Activity Issues:

• Low GTPase Activity

  • Ensure proper buffer composition (especially Mg²⁺ concentration)

  • Check protein folding using spectroscopic methods

  • Verify the presence of required co-factors

  • Test protein from different expression systems to assess post-translational modification effects

• Poor Ribosome Interaction

  • Optimize salt concentration in binding buffer

  • Ensure ribosomes are active and properly prepared

  • Check for interfering contaminants in the protein preparation

• Systematic Troubleshooting Approach:

  • Prepare a reference table comparing different expression conditions and corresponding activity measurements

  • Implement control experiments at each step

  • Compare activity of the recombinant protein to a known active reference (if available)
    When analyzing results, consider that lepA activity is inherently dependent on proper folding and may be significantly affected by the expression system chosen.

What bioinformatic approaches can be used to study B. licheniformis lepA evolution and conservation?

Bioinformatic analysis of Bacillus licheniformis Elongation Factor 4 provides valuable insights into its evolutionary history, structural conservation, and functional importance. A comprehensive bioinformatic approach should include:

• Sequence Analysis:

  • Multiple sequence alignment of lepA homologs across bacterial species

  • Identification of conserved motifs, particularly in GTP-binding domains

  • Calculation of conservation scores for each residue

• Phylogenetic Analysis:

  • Construction of phylogenetic trees using maximum likelihood or Bayesian methods

  • Comparison of lepA evolution with species evolution to identify horizontal gene transfer events

  • Analysis of selection pressure using dN/dS ratios

• Structural Prediction and Analysis:

  • Homology modeling based on solved structures of lepA homologs

  • Molecular dynamics simulations to assess structural stability

  • Prediction of functional sites using conservation mapping onto 3D structures

• Genomic Context Analysis:

  • Examination of genes flanking lepA in B. licheniformis

  • Identification of operonic structures and potential co-regulation

  • Comparative genomics across Bacillus species

• Domain Architecture Analysis:

  • Identification of domain boundaries and comparison with other translation factors

  • Analysis of domain fusion events during evolution

• Tools and Resources:

  • Sequence databases: UniProt, NCBI Protein

  • Alignment tools: MUSCLE, CLUSTAL Omega

  • Phylogenetic software: MEGA, MrBayes, RAxML

  • Structural prediction: I-TASSER, AlphaFold2

  • Visualization: PyMOL, Chimera
    This comprehensive bioinformatic approach provides a framework for understanding the evolutionary constraints on lepA and can guide experimental investigations of structure-function relationships.

How can recombinant B. licheniformis lepA be utilized in studies of translation fidelity?

Recombinant Bacillus licheniformis Elongation Factor 4 offers unique opportunities for investigating translation fidelity mechanisms due to its role as a ribosomal back-translocase. Researchers can utilize this protein in several experimental systems:

• In vitro Translation Systems:

  • Reconstituted translation systems supplemented with purified recombinant lepA (>85% purity)

  • Measurement of misincorporation rates using reporter constructs with known error-prone codons

  • Analysis of lepA's effect on readthrough of premature termination codons

• Single-Molecule Studies:

  • FRET-based assays to directly visualize lepA-mediated ribosomal movement

  • Optical tweezers experiments to measure the force generated during back-translocation

• Error-Induction Models:

  • Systems with elevated error rates induced by antibiotics or other stressors

  • Assessment of how lepA supplementation affects error correction

• Comparative Studies:

  • Side-by-side comparison of lepA from B. licheniformis with homologs from other species

  • Structural basis for any observed functional differences
    This research direction is particularly valuable for understanding fundamental translation mechanisms and potentially developing new approaches to modulate translation fidelity in biotechnological applications.

What is the potential relationship between B. licheniformis lepA and the bacterium's probiotic properties?

While the direct relationship between Bacillus licheniformis Elongation Factor 4 and the organism's probiotic properties has not been specifically established in the search results, several hypothetical connections warrant investigation:

• Stress Adaptation Mechanism:

  • B. licheniformis demonstrates protective effects against LPS-induced stress in animal models

  • LepA's role in maintaining translation fidelity under stress conditions may contribute to the bacterium's resilience in challenging gut environments

• Protein Quality Control:

  • Probiotic effects often depend on properly folded bacterial proteins interacting with host systems

  • LepA's function in translation quality control could ensure proper synthesis of proteins involved in:

    • Immunomodulation (affecting cytokines like IL-10, TNF-α, IL-6)

    • Antioxidant activities (improving GSH-Px, SOD and T-AOC activities)

    • Exopolysaccharide production (which demonstrates antioxidant and antibacterial activities)

• Research Approaches to Explore This Connection:

  • Comparative transcriptomics of wild-type vs. lepA-deficient B. licheniformis under probiotic-relevant conditions

  • Animal studies using lepA knockout strains to assess impact on probiotic properties

  • Analysis of protein synthesis fidelity under gut-relevant stress conditions
    This potential connection represents an intriguing area for future research that could elucidate molecular mechanisms underlying probiotic effects.

How might advances in B. licheniformis lepA research contribute to biotechnology applications?

Research on Bacillus licheniformis Elongation Factor 4 has several potential biotechnological applications:

• Protein Engineering Platform:

  • Development of engineered lepA variants with enhanced back-translocation activity

  • Creation of chimeric translation factors with novel functions

  • Design of lepA-based tools for controlling translation fidelity in biotechnological processes

• Biopharmaceutical Production Enhancement:

  • Integration with expression systems to improve recombinant protein quality

  • Potential co-expression with therapeutic proteins to enhance folding or reduce truncation products

  • Implementation in cell-free protein synthesis systems for production of difficult-to-express proteins

• Antibiotic Development:

  • Use of structural information about B. licheniformis lepA to design novel antibiotics targeting this essential translation factor

  • Screening for compounds that specifically modulate lepA activity

• Probiotic Enhancement:

  • Engineering B. licheniformis strains with optimized lepA expression for enhanced stress resistance

  • Development of next-generation probiotics with improved therapeutic properties

• Diagnostic Tools:

  • Creation of lepA-based biosensors for detecting translation stress

  • Development of screening platforms for compounds affecting translation fidelity These applications build upon the fundamental understanding of lepA's structure and function, potentially leading to innovative biotechnological tools and products.