Recombinant Lactobacillus plantarum Elongation factor 4 2 (lepA2), partial

What is Lactobacillus plantarum Elongation factor 4 2 (lepA2) and what are its primary functions?

Lactobacillus plantarum Elongation factor 4 2 (lepA2) is a protein classified as a ribosomal back-translocase (EC 3.6.5.n1). It functions primarily in the translational machinery of L. plantarum, facilitating protein synthesis by catalyzing the back-translocation of ribosomes along mRNA. This process is crucial for ensuring translation fidelity. The protein is also known as EF-4 2 or Ribosomal back-translocase LepA 2, and has been identified in L. plantarum strain ATCC BAA-793/NCIMB 8826/WCFS1 with UniProt accession number Q88T65 .

Methodologically, researchers studying lepA2 function should employ ribosome profiling techniques coupled with translation efficiency assays to understand its role in protein synthesis accuracy. Comparative studies with lepA2-deficient strains can reveal its specific contributions to cellular physiology and stress responses in L. plantarum.

How does the structure of lepA2 compare to other elongation factors in bacteria?

The lepA2 protein shares structural similarities with other bacterial elongation factors, particularly EF-G, but contains unique domains that distinguish its function. While EF-G promotes forward translocation during elongation, lepA2 (EF-4) can reverse this movement under specific conditions, acting as a "molecular brake" in translation.

To study these structural differences methodologically:

• Perform sequence alignment analysis using tools like CLUSTAL_X with other bacterial elongation factors

• Generate structural models using X-ray crystallography or cryo-EM

• Conduct domain-specific functional studies using site-directed mutagenesis to identify critical regions for back-translocation activity

Comparison studies have shown that while the core GTPase domain is conserved among elongation factors, lepA2 contains unique C-terminal regions that are essential for its specialized function in ribosome dynamics and translational fidelity.

What are the optimal conditions for expressing recombinant lepA2 in heterologous systems?

For optimal expression of recombinant lepA2, researchers should consider the following methodological approach:

• Expression system selection: Baculovirus expression systems have been successfully used for lepA2 expression . Alternatively, E. coli-based systems with codon optimization for L. plantarum sequences can be employed.

• Optimal expression conditions:

  • Temperature: 37°C appears optimal for protein expression

  • Induction time: 6-10 hours (based on similar recombinant protein expression studies for L. plantarum)

  • pH: Neutral (7.0-7.5)

  • Media: Rich media supplemented with appropriate antibiotics

• Purification strategy:

  • Initial centrifugation at 8,000 r/min for 10 minutes to collect bacterial cells

  • Lysis under native conditions

  • Affinity chromatography (typically His-tag based)

  • Size exclusion chromatography for higher purity

The resulting protein should achieve >85% purity as determined by SDS-PAGE analysis . For activity preservation, adding 5-50% glycerol to the final preparation is recommended, with 50% being the standard concentration for long-term storage.

What methods are most effective for assessing the functional activity of recombinant lepA2?

To effectively assess the functional activity of recombinant lepA2, researchers should employ a multi-faceted approach:

• GTPase activity assay:

  • Measure GTP hydrolysis rates using malachite green phosphate detection

  • Compare activity with and without ribosomes to determine ribosome-dependent stimulation

• Ribosome binding assays:

  • Use filter binding techniques with radiolabeled GTP

  • Employ microscale thermophoresis to measure binding affinities

• In vitro translation fidelity assays:

  • Reconstituted translation systems with reporter constructs

  • Measure misincorporation rates and frameshifting frequencies

• Biochemical characterization:

  • Temperature stability testing (stable up to 50°C according to similar L. plantarum proteins)

  • pH stability (stable at pH as low as 1.5)

  • Salt resistance testing

A comprehensive evaluation should include control experiments with known elongation factors (EF-Tu, EF-G) for comparison. When interpreting results, consider that lepA2 activity is often condition-dependent, becoming more significant under stress conditions that affect translational fidelity.

How can lepA2 be utilized in studies of L. plantarum adaptation to different gut environments?

LepA2 can serve as a molecular marker for studying L. plantarum adaptation to gut environments through the following methodological approaches:

• Comparative expression analysis:

  • Quantify lepA2 expression levels in L. plantarum isolated from different gut environments using RT-qPCR

  • Correlate expression patterns with environmental stressors (pH, bile concentration, nutrient availability)

• Mutant survival studies:

  • Generate lepA2 knockout or overexpression strains

  • Assess their colonization efficiency in gut models compared to wild-type strains

  • Measure competitive fitness through in vivo competition assays

• Host interaction analysis:

  • Evaluate the impact of lepA2 expression on immune response modulation

  • Assess adhesion properties to intestinal epithelial cells

  • Measure inflammatory marker production (TNF-α, IL-6) in response to different lepA2 expression levels

Research has shown that L. plantarum adaptability to gut environments involves complex regulation of translation machinery components. For example, a study demonstrated that L. plantarum isolated from the gut of mice supplemented with different dietary components showed significant differences in gene expression patterns, including those involved in environmental information processing . Similar approaches can be applied to understand lepA2's role in adaptation.

What is the role of lepA2 in L. plantarum stress responses and how can this be experimentally determined?

The role of lepA2 in L. plantarum stress responses can be methodologically investigated through:

• Stress exposure experiments:

  • Subject L. plantarum cultures to various stressors (acid, bile, oxidative, osmotic, heat)

  • Quantify lepA2 expression changes using RT-qPCR or RNA-seq

  • Compare with other stress response genes to establish correlation patterns

• Genetic manipulation approaches:

  • Create lepA2 deletion mutants and assess their survival under stress conditions

  • Complement with wild-type lepA2 to confirm phenotype restoration

  • Develop lepA2-reporter fusion constructs to monitor real-time expression

• Proteomic analysis:

  • Compare proteome profiles of wild-type and lepA2-deficient strains under stress

  • Identify differentially expressed proteins in stress response pathways

  • Use techniques like 2D-DIGE or LC-MS/MS for comprehensive protein profiling

• Translational fidelity assessment:

  • Measure mistranslation rates under stress conditions in wild-type vs. lepA2 mutants

  • Use reporter constructs with programmed frameshifting or stop codon readthrough sites

Research suggests that translation factors like lepA2 play crucial roles during stress adaptation by maintaining protein synthesis fidelity when translation machinery is compromised. For instance, studies on other Lactobacillus species have demonstrated significant changes in translational apparatus components during acid or bile stress adaptation, indicating a potential role for lepA2 in these processes .

What are the most common challenges in purifying active recombinant lepA2 and how can they be overcome?

Common challenges in purifying active recombinant lepA2 and their methodological solutions include:

• Low expression levels:

  • Optimize codon usage for the expression host

  • Test different promoter systems (T7, tac, araBAD)

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

  • Consider using specialized expression strains (e.g., BL21-CodonPlus for E. coli)

• Protein insolubility/inclusion body formation:

  • Lower expression temperature (16-25°C)

  • Co-express with molecular chaperones (GroEL/ES, DnaK/J)

  • Use solubility-enhancing fusion tags (MBP, SUMO, TrxA)

  • Optimize lysis buffer composition with mild detergents

• Maintaining enzymatic activity:

  • Include stabilizing agents during purification (glycerol, reducing agents)

  • Minimize freeze-thaw cycles (aliquot and store at -80°C)

  • Add protease inhibitors throughout purification

  • Consider native purification conditions

• Contamination with host proteins:

  • Implement a multi-step purification strategy (affinity, ion exchange, size exclusion)

  • Include more stringent washing steps during affinity purification

  • Consider on-column refolding for proteins purified from inclusion bodies

According to product specifications, recombinant lepA2 preparations can achieve >85% purity using appropriate purification techniques . For optimal stability, storage at -20°C/-80°C with 50% glycerol is recommended, with shelf life of approximately 6 months for liquid form and 12 months for lyophilized preparations. Repeated freezing and thawing should be avoided, and working aliquots can be stored at 4°C for up to one week .

How can researchers address potential contamination issues when working with recombinant proteins from L. plantarum?

To address contamination issues when working with recombinant proteins from L. plantarum, researchers should implement the following methodological approaches:

• Source material verification:

  • Confirm L. plantarum strain identity through 16S rRNA sequencing

  • Use validated strains (e.g., ATCC BAA-793/NCIMB 8826/WCFS1)

  • Perform regular purity checks of bacterial cultures

• Expression system considerations:

  • When using heterologous systems like Baculovirus , implement regular screening for adventitious agents

  • For bacterial expression systems, use well-characterized laboratory strains with defined genetic backgrounds

  • Consider dedicated equipment to prevent cross-contamination

• Purification strategy:

  • Include multiple orthogonal purification steps

  • Implement endotoxin removal procedures (especially important for proteins intended for immunological studies)

  • Use sterile filtration (0.22 μm) as a final step

• Quality control methods:

  • SDS-PAGE with silver staining to detect low-level contaminants

  • Mass spectrometry to confirm protein identity and purity

  • Activity assays to ensure functional integrity

  • Endotoxin testing using LAL assay for preparations used in cell culture

• Microbial contamination prevention:

  • Use aseptic technique throughout processing

  • Prepare buffers with sterile, endotoxin-free water

  • Implement regular environmental monitoring in the laboratory

For specific applications like immunological studies or when studying L. plantarum-host interactions , additional steps such as host cell protein (HCP) analysis and removal of lipopolysaccharides may be necessary to ensure experimental results are not confounded by contaminants.

How might lepA2 contribute to the probiotic properties of L. plantarum and what experimental approaches could test this hypothesis?

To investigate lepA2's potential contribution to L. plantarum's probiotic properties, researchers can employ these methodological approaches:

• Gene expression correlation studies:

  • Compare lepA2 expression levels across L. plantarum strains with varying probiotic efficacy

  • Analyze lepA2 expression changes during host colonization or stress conditions

  • Correlate expression with specific probiotic traits (e.g., acid resistance, adhesion capability)

• Genetic modification experiments:

  • Generate lepA2 knockout, knockdown, and overexpression strains

  • Assess these mutants for probiotic characteristics:

    • Acid and bile tolerance using survival assays at pH 1.0-5.0 and 0.3-2.0% bile salts

    • Adhesion properties using bacterial adhesion to hydrocarbons (BATH) assay with various hydrocarbons (xylene, chloroform, n-hexadecane)

    • Auto-aggregation and co-aggregation properties with pathogens

    • Antimicrobial activity against pathogens like E. coli, S. aureus, and B. cereus

• Translational efficiency assessment:

  • Measure protein synthesis rates under stress conditions in wild-type vs. lepA2 mutants

  • Analyze the impact on expression of key probiotic-associated proteins

• In vivo testing:

  • Compare the efficacy of wild-type and lepA2-modified strains in animal models

  • Assess parameters like:

    • Colonization efficiency in intestinal tract

    • Protective effects against pathogen challenges

    • Immunomodulatory effects, such as changes in inflammatory markers

    • Effects on host physiology (e.g., hyperuricemia models)

Research has shown that L. plantarum strains possess various probiotic attributes including adhesion properties (with auto-aggregation percentages reaching 97-98% after 5 hours of incubation), antimicrobial activity against pathogens, and tolerance to acid and bile conditions . The relationship between lepA2 function and these properties remains to be elucidated but may involve translational regulation during stress adaptation.

What comparative genomic approaches could reveal about the evolution and functional diversification of lepA2 across Lactobacillus species?

To investigate lepA2's evolution and functional diversification across Lactobacillus species, researchers should consider these methodological approaches:

• Sequence acquisition and alignment:

  • Collect lepA2 sequences from diverse Lactobacillus species and strains

  • Perform multiple sequence alignment using tools like CLUSTAL_X

  • Calculate sequence conservation, identifying conserved and variable regions

• Phylogenetic analysis:

  • Construct phylogenetic trees using methods like neighbor-joining

  • Implement tools such as MEGA-6.0 for tree construction and visualization

  • Compare lepA2 phylogeny with species phylogeny to identify potential horizontal gene transfer events

• Structural prediction and comparison:

  • Generate structural models of lepA2 from different species

  • Identify structurally conserved domains vs. variable regions

  • Correlate structural differences with habitat adaptations or functional specialization

• Functional domain analysis:

  • Map functional domains across species

  • Identify species-specific insertions/deletions or substitutions

  • Correlate domain variations with known ecological niches or metabolic capabilities

• Selection pressure analysis:

  • Calculate Ka/Ks ratios to identify regions under positive or purifying selection

  • Identify potential adaptive mutations in specific lineages

  • Correlate selection patterns with bacterial lifestyle differences

Research has shown that Lactobacillus species demonstrate considerable genomic diversity, including variations in translation-related genes. For example, studies on L. salivarius revealed that while chromosomal genes tend to be more conserved, plasmid-encoded genes show greater variation . Similar approaches can be applied to understand lepA2 evolution. The functional implications of lepA2 variants could be tested through complementation studies, where lepA2 genes from different species are expressed in a common host background to assess functional equivalence or specialization.

How should researchers interpret seemingly contradictory data regarding lepA2 function in different experimental contexts?

When faced with contradictory data regarding lepA2 function, researchers should employ the following methodological framework:

• Systematic variation analysis:

  • Catalog all experimental variables across studies (strain backgrounds, growth conditions, assay methods)

  • Create a correlation matrix between variables and outcomes

  • Identify potential confounding factors using statistical approaches

• Reproducibility assessment:

  • Replicate key experiments under identical conditions

  • Implement blinded analysis to reduce bias

  • Consider inter-laboratory validation for critical findings

• Strain-specific effects investigation:

  • Compare lepA2 sequence and expression across different L. plantarum strains

  • Test multiple reference strains (e.g., ATCC BAA-793, clinical isolates, food-derived strains)

  • Consider genomic context differences that might influence lepA2 function

• Condition-dependent functionality analysis:

  • Systematically vary experimental conditions (pH, temperature, nutrient availability)

  • Implement factorial design to identify interaction effects

  • Test lepA2 function under physiologically relevant stress conditions

• Molecular mechanism reconciliation:

  • Develop mechanistic models that could explain seemingly contradictory results

  • Design critical experiments to differentiate between competing models

  • Consider potential post-translational modifications or interaction partners

Research on L. plantarum has shown strain-specific differences in functionality even within the same species. For example, studies have demonstrated that L. plantarum strains can exhibit different protective effects in disease models, with strain Lp2 showing significant improvement in LPS-induced liver injury , while other strains demonstrate different metabolic capabilities or stress responses . The same methodological approach can be applied to understand context-dependent lepA2 function.

What biostatistical approaches are most appropriate for analyzing lepA2 expression data across different experimental conditions?

For robust analysis of lepA2 expression data across different experimental conditions, researchers should apply these biostatistical methodologies:

• Experimental design optimization:

  • Implement power analysis to determine appropriate sample sizes

  • Use randomized block designs to control for batch effects

  • Include appropriate technical and biological replicates (minimum n=3 per condition)

• Data normalization strategies:

  • For RT-qPCR: Use multiple reference genes validated for stability under experimental conditions

  • For RNA-seq: Apply appropriate normalization methods (TPM, RPKM, or DESeq2 normalization)

  • Consider global vs. local normalization approaches based on experimental context

• Statistical testing framework:

  • For comparing two conditions: t-tests with appropriate corrections for multiple testing

  • For multiple conditions: ANOVA followed by post-hoc tests (e.g., Duncan's post-hoc test)

  • For time-series data: Consider repeated measures ANOVA or mixed-effects models

• Advanced analytical approaches:

  • Principal Component Analysis (PCA) for identifying major sources of variation

  • Partial Least Squares-Discriminant Analysis (PLS-DA) for group discrimination

  • Hierarchical clustering to identify co-regulated genes

  • Correlation analysis to identify genes with similar expression patterns to lepA2

• Visualization techniques:

  • Heat maps for displaying expression patterns across multiple conditions

  • Volcano plots to visualize significance vs. fold change

  • Box plots with individual data points to show distribution and outliers

When analyzing lepA2 expression data, special consideration should be given to context-dependent variables. For example, when examining L. plantarum in different host environments, researchers have employed specialized statistical approaches such as Spearman correlation analysis to identify relationships between bacterial abundance and host parameters . The statistical significance threshold is typically set at p < 0.05, with appropriate corrections for multiple comparisons.

How can lepA2 be used to study L. plantarum adaptation mechanisms in the mammalian gut environment?

To use lepA2 as a tool for studying L. plantarum adaptation in the mammalian gut, researchers should implement these methodological approaches:

• In vivo expression profiling:

  • Isolate L. plantarum from different gut regions (small intestine, cecum, colon)

  • Perform RT-qPCR or RNA-seq to measure lepA2 expression levels

  • Compare gut expression to in vitro growth conditions to identify gut-specific regulation

• Genetic reporter systems:

  • Construct lepA2 promoter-reporter fusions (GFP, luciferase)

  • Generate L. plantarum strains carrying these constructs

  • Monitor real-time expression in gut models or transparent animal models

• Host-induced adaptation experiments:

  • Subject L. plantarum to gut-like conditions (low pH, bile acids, nutrient limitation)

  • Measure lepA2 expression changes over time

  • Correlate with adaptive phenotypes (stress resistance, biofilm formation)

• Comparative analysis across host species:

  • Isolate L. plantarum from different host species (humans, mice, geese)

  • Compare lepA2 sequence and expression patterns

  • Identify host-specific adaptations in regulatory mechanisms

Research has demonstrated that L. plantarum strains show different adaptation strategies in response to host dietary supplements. For example, whole genome resequencing and intracellular metabolite detection revealed that dietary carbohydrates, peptides, and minerals have distinct effects on L. plantarum gene expression in the gut of mice, particularly affecting environmental information processing pathways . Similar approaches can be applied to understand lepA2's role in gut adaptation.

What role might lepA2 play in L. plantarum's therapeutic applications and how can this be experimentally validated?

To investigate lepA2's potential role in L. plantarum's therapeutic applications, researchers should employ these methodological approaches:

• Expression correlation in therapeutic models:

  • Compare lepA2 expression levels between therapeutic and non-therapeutic strains

  • Measure expression changes during therapeutic applications (e.g., liver injury treatment , hyperuricemia amelioration )

  • Identify regulatory patterns specific to therapeutic outcomes

• Genetic modification validation:

  • Generate lepA2 knockout and overexpression strains

  • Test their therapeutic efficacy in disease models compared to wild-type strains

  • Assess parameters like:

    • Inflammatory markers (TNF-α, IL-6, IL-1β)

    • Biochemical indicators of disease (ALT, AST, serum uric acid)

    • Histopathological outcomes

• Mechanism elucidation studies:

  • Investigate lepA2's impact on translational regulation of therapeutic proteins

  • Analyze proteome changes in wild-type vs. lepA2 mutants during therapeutic applications

  • Identify potential lepA2-dependent pathways contributing to therapeutic effects

• Host response analysis:

  • Compare immune responses to wild-type vs. lepA2-modified strains

  • Profile host gene expression changes using microarray or RNA-seq

  • Correlate with therapeutic outcomes

Research has demonstrated various therapeutic applications of L. plantarum, including improvement of LPS-induced liver injury through the TLR-4/MAPK/NFκB and Nrf2-HO-1/CYP2E1 pathways , alleviation of hyperuricemia by promoting nucleoside uptake and hydrolysis , and prevention of severe pathogenesis in leptospirosis models . Understanding lepA2's contribution to these effects could enable the development of more effective probiotic formulations through rational strain engineering.

A systematic approach analyzing lepA2 expression across different therapeutic models, coupled with phenotypic assessment of lepA2-modified strains, would provide valuable insights into its potential role in probiotic therapies.

What emerging technologies might advance our understanding of lepA2 function in L. plantarum?

Several emerging technologies hold promise for advancing our understanding of lepA2 function in L. plantarum:

• CRISPR-Cas9 genome editing:

  • Enable precise modification of lepA2 sequences

  • Create conditional expression systems and domain-specific mutations

  • Implement CRISPRi for temporal regulation of lepA2 expression

• Single-cell technologies:

  • Apply single-cell RNA-seq to identify cell-to-cell variation in lepA2 expression

  • Use microfluidics to analyze cellular responses at the individual level

  • Implement time-lapse microscopy with reporter constructs to track lepA2 expression dynamics

• Ribosome profiling (Ribo-seq):

  • Map ribosome positioning genome-wide in wildtype vs. lepA2 mutants

  • Identify specific mRNAs affected by lepA2 activity

  • Quantify translational efficiency changes under various conditions

• Structural biology advances:

  • Apply cryo-EM to visualize lepA2-ribosome interactions

  • Use hydrogen-deuterium exchange mass spectrometry to map conformational changes

  • Implement computational modeling to predict functional interactions

• High-throughput phenotyping:

  • Employ Biolog phenotype microarrays to assess metabolic consequences of lepA2 modification

  • Use TraDIS or similar approaches to identify genetic interactions with lepA2

  • Implement automated growth analysis under diverse conditions

These technologies would allow researchers to move beyond correlative observations to mechanistic understanding of lepA2 function. For example, while studies have identified associations between L. plantarum strains and therapeutic outcomes , emerging technologies could elucidate the specific contributions of lepA2 to these complex phenotypes.

How might systematic characterization of lepA2 variants contribute to improved probiotic and therapeutic applications of L. plantarum?

Systematic characterization of lepA2 variants could significantly advance probiotic and therapeutic applications of L. plantarum through these methodological approaches:

• Natural variant cataloging:

  • Sequence lepA2 across diverse L. plantarum isolates from different niches

  • Correlate sequence variations with source environments and strain properties

  • Identify potential adaptive mutations associated with specific functions

• Structure-function relationship mapping:

  • Generate a library of lepA2 variants with defined mutations

  • Assess their impact on protein function and bacterial phenotype

  • Identify key residues or domains associated with specific probiotic traits

• Rational strain engineering:

  • Introduce beneficial lepA2 variants into probiotic strains

  • Optimize expression levels for specific applications

  • Create strains with enhanced stability or functionality in target environments

• Application-specific optimization:

  • Identify lepA2 variants that enhance particular therapeutic applications:

    • Improved hyperuricemia amelioration

    • Enhanced liver protection capabilities

    • Better immune modulation properties

  • Validate enhanced efficacy in appropriate disease models

• Safety and stability assessment:

  • Evaluate potential off-target effects of lepA2 modifications

  • Assess stability and persistence of engineered strains

  • Ensure modified strains maintain probiotic properties like acid/bile tolerance and adherence capacity

Research has demonstrated that L. plantarum strains can be engineered for enhanced functionality, as seen in the development of recombinant strains expressing coronavirus spike protein . Similar approaches could be applied to optimize lepA2 for improved translational regulation under specific conditions, potentially enhancing the therapeutic efficacy of L. plantarum in applications ranging from metabolic disorders to immune modulation.