Recombinant Lactobacillus johnsonii UPF0398 protein LJ_1195 (LJ_1195)

What is Lactobacillus johnsonii UPF0398 protein LJ_1195 and how is it characterized?

Lactobacillus johnsonii UPF0398 protein LJ_1195 is a protein encoded by the LJ_1195 gene in the L. johnsonii genome. It belongs to the UPF0398 protein family, a group of uncharacterized proteins with conserved domains across various bacterial species. The protein appears in the L. johnsonii genome sequence documentation and has been identified in genomic analyses .

The protein characterization typically involves:

• Molecular weight determination: Approximately 30-40 kDa based on related protein data

• Sequence analysis and comparison with homologous proteins

• Structural prediction using bioinformatics tools

• Functional analysis through recombinant expression and purification

What approaches are recommended for expressing recombinant L. johnsonii proteins?

Several expression systems have demonstrated effectiveness for L. johnsonii proteins:

• Yeast expression systems: Commonly used for proteins requiring eukaryotic post-translational modifications .

• Baculovirus expression system: Effective for proteins that may be difficult to express in bacterial systems, particularly when higher yields are required .

• E. coli expression systems: Using vectors such as pET or pGEX for high-yield expression with appropriate codon optimization.

• Lactobacillus expression systems: When native conformation is critical, homologous expression in Lactobacillus species preserves natural folding environments.

Methodology considerations include:

• Codon optimization for the host organism

• Selection of appropriate promoters (constitutive vs. inducible)

• Inclusion of purification tags (His-tag, GST)

• Growth conditions optimization (temperature, induction timing)

How does L. johnsonii genomic context inform protein function studies?

The genomic context of L. johnsonii provides critical insights for protein function studies:

L. johnsonii has a genome size of approximately 1.88 million base pairs with a GC content of 34.4% . The genome contains approximately 1,865 genes, including 1,772 protein-coding genes . Understanding this genomic landscape helps researchers:

• Identify potential operons containing LJ_1195

• Evaluate gene expression patterns through transcriptomic data

• Assess evolutionary conservation across Lactobacillus species

• Predict functional roles based on genomic neighborhood

Recent phylogenetic analyses show L. johnsonii strain MT4 is closely related to strain NCK2677 (>99.96% genome identity) and NCC 533, a strain with documented probiotic properties .

What methodological approaches are optimal for studying LJ_1195 protein interactions with host proteins?

To investigate interactions between LJ_1195 and host proteins, researchers should consider multiple complementary approaches:

• Affinity purification coupled to mass spectrometry:

  • Expressing tagged LJ_1195 and identifying binding partners

  • Reciprocal verification using identified partner proteins as bait

  • Cross-linking prior to purification to capture transient interactions

• Surface plasmon resonance (SPR):

  • Quantifying binding kinetics between purified LJ_1195 and candidate host proteins

  • Determining association/dissociation constants

• Co-immunoprecipitation from biological samples:

  • Using antibodies against LJ_1195 to pull down protein complexes

  • Western blot verification of specific interactions

• Functional assays:

  • Based on the GAPDH-JAM-2 interaction model demonstrated for other L. johnsonii proteins

  • Cell-based assays measuring barrier function in epithelial models

The L. johnsonii GAPDH-JAM-2 interaction model provides a valuable precedent, as it was shown to enhance gut barrier integrity via binding between bacterial GAPDH and host JAM-2 tight junction protein .

How might LJ_1195 contribute to the oxidative stress response in L. johnsonii?

L. johnsonii has a complex relationship with oxygen and oxidative stress that may involve LJ_1195:

• Oxidative stress physiology:

  • L. johnsonii produces H₂O₂ via NADH dependent flavin reductase and NADH oxidase activities

  • The strain possesses genes encoding products with potential antioxidant functions

• Experimental approaches to study LJ_1195 role:

  • Gene knockout/knockdown studies with subsequent oxidative challenge tests

  • Protein activity assays under varying oxygen concentrations

  • Transcriptome analysis comparing wildtype and mutant strains under oxidative stress

• Potential mechanisms:

  • May function in electron transport pathways

  • Could play a role in NADH oxidation similar to other oxidoreductases

  • Might contribute to maintaining redox balance

Research has shown that oxygen exposure relieves CO₂ and acetate dependency in L. johnsonii NCC 533 , suggesting complex metabolic adaptations to aerobic conditions that might involve proteins like LJ_1195.

What transcriptomic approaches reveal insights into LJ_1195 expression patterns?

Transcriptomic analysis provides valuable insights into the expression patterns of LJ_1195:

• RNA-Seq methodology:

  • Total RNA extraction from L. johnsonii under various conditions

  • Library preparation with rRNA depletion

  • Next-generation sequencing with >10 million reads per sample

  • Bioinformatic analysis with tools like DESeq2 for differential expression

• Experimental conditions to consider:

  • Growth phase variations (lag, log, stationary)

  • Environmental stressors (pH, temperature, oxygen levels)

  • Nutrient availability variations

  • Host-mimicking conditions (intestinal environment)

• Data interpretation frameworks:

  • Co-expression network analysis to identify functional relationships

  • Pathway enrichment analysis for biological context

  • Comparison with proteomics data for validation

Research on L. johnsonii has demonstrated that CO₂ depletion triggers significant transcriptional changes, including upregulation of pyrimidine synthesis pathways . Similar approaches can be applied to study LJ_1195 expression patterns.

How can functional genomics approaches elucidate the role of LJ_1195 in bacterium-host interactions?

Functional genomics offers multiple strategies to understand LJ_1195's potential role in host interactions:

• Gene knockout/knockdown approaches:

  • CRISPR-Cas9 system adapted for Lactobacillus

  • Homologous recombination for gene deletion

  • Antisense RNA for expression reduction

• Heterologous expression systems:

  • Expression in standard laboratory strains with specific phenotypic readouts

  • Complementation studies to verify function

• In vitro models:

  • Caco-2 cell monolayers to assess barrier function impacts

  • Dendritic cell stimulation to evaluate immunomodulatory effects

  • Intestinal organoid models for complex interaction studies

• In vivo approaches:

  • Gnotobiotic mouse models colonized with wildtype or LJ_1195 mutants

  • Assessment of colonization efficiency, immune response, and barrier function

Research on L. johnsonii has demonstrated its ability to enhance gut barrier integrity through protein-protein interactions and shows promise as a mucosal vaccine delivery vehicle , suggesting complex bacterium-host interfaces that may involve UPF0398 family proteins.

What purification protocols maximize yield and activity of recombinant LJ_1195?

Purification of recombinant LJ_1195 requires carefully optimized protocols:

• Expression system selection:

  • Yeast systems for glycosylated variants

  • Bacterial systems for high yield

  • Consideration of fusion tags (His, GST, MBP) for solubility enhancement

• Lysis optimization:

  • Buffer composition (pH 7.0-8.0, 150-300 mM NaCl)

  • Protease inhibitor cocktail inclusion

  • Mechanical disruption methods for complete lysis

• Purification strategies:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged constructs

  • Size exclusion chromatography for final polishing

  • Tag removal considerations using specific proteases

• Activity preservation:

  • Addition of stabilizing agents (glycerol 10-20%)

  • Antioxidant inclusion if oxygen-sensitive

  • Storage optimization (-80°C with flash freezing)

Typical yield expectations range from 2-10 mg/L culture volume depending on expression system and optimization level.

How can structural biology approaches characterize LJ_1195 function?

Structural characterization provides crucial insights into protein function:

• Crystallography approach:

  • Protein crystallization screening (hanging drop, sitting drop)

  • X-ray diffraction analysis

  • Structural determination and refinement

• NMR spectroscopy:

  • Solution structure determination

  • Ligand binding studies

  • Dynamic interaction mapping

• Cryo-electron microscopy:

  • Single particle analysis for larger complexes

  • Structural determination without crystallization

• Computational approaches:

  • Homology modeling based on related UPF0398 family proteins

  • Molecular dynamics simulations to predict functional movements

  • Docking studies to predict interaction partners

The UPF0398 protein family structural characterization would provide insights into potential enzymatic activities, binding interfaces, and functional domains.

How might LJ_1195 contribute to L. johnsonii's probiotic properties?

LJ_1195 may play several roles in the probiotic functionality of L. johnsonii:

• Potential contribution mechanisms:

  • Antimicrobial activity against pathogens

  • Host immune modulation

  • Metabolic functions supporting colonization

  • Stress response enabling gut persistence

• Research evidence context:

  • L. johnsonii shows antagonistic relationships with pathogens like C. albicans

  • The strain demonstrates immunomodulatory properties by altering macrophage, T-cell, and Th2 cytokine levels

  • Specific L. johnsonii strains (N6.2) have been shown to mitigate type 1 diabetes onset in animal models

• Experimental approaches:

  • Comparison of wildtype and LJ_1195 mutant strains in probiotic function assays

  • Recombinant protein administration studies

  • Transcriptomic analysis of host response to purified LJ_1195

L. johnsonii has been documented to enhance gut barrier integrity through protein-protein interactions , suggesting specialized molecular mechanisms behind its probiotic effects.

What methodologies can assess LJ_1195's role in interspecies microbial interactions?

Several methods can evaluate LJ_1195's potential role in microbial ecology:

• Co-culture experiments:

  • Growth of L. johnsonii (wildtype vs. LJ_1195 mutants) with other microbes

  • Measurement of growth impact, metabolite exchange, and gene expression changes

  • Assessment of antimicrobial compound production

• Metagenomic/metatranscriptomic approaches:

  • Analysis of microbial community changes in response to L. johnsonii colonization

  • Comparative studies with wildtype versus LJ_1195 mutants

• Biofilm models:

  • Multi-species biofilm formation analysis

  • Confocal microscopy to visualize spatial relationships

  • Quantification of biofilm parameters

Research demonstrates that L. johnsonii has an antagonistic relationship with C. albicans during both planktonic and biofilm growth, with significant reductions in biofilm parameters when L. johnsonii is present :

These findings suggest L. johnsonii proteins may play important roles in interspecies interactions.

How can systems biology integrate LJ_1195 function into broader L. johnsonii metabolic networks?

Systems biology approaches offer comprehensive frameworks for understanding LJ_1195 in context:

• Genome-scale metabolic modeling:

  • Integration of LJ_1195 into existing L. johnsonii metabolic models

  • Flux balance analysis to predict metabolic impacts

  • Simulation of gene knockout effects on growth and metabolite production

• Multi-omics data integration:

  • Correlation of transcriptomics, proteomics, and metabolomics data

  • Network analysis to identify functional modules

  • Identification of condition-specific regulatory patterns

• Experimental validation approaches:

  • Metabolic flux analysis using isotope labeling

  • Targeted metabolomics to verify predicted changes

  • Growth phenotyping under various nutrient conditions

L. johnsonii has complex nutrient requirements and metabolic adaptations, including CO₂-dependent pyrimidine synthesis pathways and the ability to utilize specific fatty acids like erucic acid , providing context for understanding specialized protein functions within its metabolic network.

What controls and validation steps are essential when working with recombinant LJ_1195?

Rigorous controls and validation steps ensure reliable research outcomes:

• Expression validation:

  • Western blot confirmation using antibodies against the target or tag

  • Mass spectrometry verification of protein identity

  • Activity assays if function is known

• Purification quality controls:

  • SDS-PAGE with Coomassie staining (>95% purity standard)

  • Endotoxin testing for preparations used in immunological studies

  • Aggregation assessment via dynamic light scattering

• Functional validation approaches:

  • Comparison with native protein where possible

  • Dose-response relationships in functional assays

  • Multiple biological replicates (minimum n=3)

  • Independent verification using different expression/purification batches

• Negative controls in experiments:

  • Heat-inactivated protein preparations

  • Unrelated proteins of similar size

  • Empty vector controls in expression studies

When designing deletion mutants for functional studies, complementation experiments are critical to confirm phenotypes are specifically due to LJ_1195 absence.

How can researchers address potential contradictions in LJ_1195 functional data?

Addressing contradictory results requires systematic troubleshooting:

• Common sources of contradictions:

  • Strain-specific variations in Lactobacillus johnsonii

  • Different experimental conditions (temperature, pH, media composition)

  • Variations in protein preparation methods

  • Host cell or animal model differences

• Resolution strategies:

  • Side-by-side comparison of contradictory protocols

  • Systematic variation of individual parameters

  • Collaboration with laboratories reporting different results

  • Meta-analysis of published data with attention to methodological details

• Experimental design approach:

  • Multiple complementary methods to test the same hypothesis

  • Inclusion of positive and negative controls in each experiment

  • Blind analysis of results when possible

  • Pre-registration of experimental plans and analysis strategies

The research on L. johnsonii has shown strain-specific variations, with different strains like MT4, NCK2677, and NCC 533 showing distinct genetic and functional characteristics despite high genomic similarity , highlighting the importance of strain-specific considerations.

What are the optimal conditions for studying LJ_1195 interactions with host immune components?

Creating physiologically relevant conditions is critical for immune interaction studies:

• Cell culture considerations:

  • Primary immune cells vs. cell lines (advantages/limitations)

  • Culture conditions mimicking intestinal environment

  • Oxygen levels (hypoxic conditions may be more relevant)

  • Presence of appropriate growth factors and cytokines

• Protein preparation factors:

  • Endotoxin removal (<0.1 EU/ml standard)

  • Proper folding verification

  • Concentration ranges spanning physiological levels

• Readout selection:

  • Cytokine production (ELISA, multiplex assays)

  • Cell surface marker expression (flow cytometry)

  • Gene expression analysis (RT-qPCR, RNA-Seq)

  • Functional assays (phagocytosis, migration, maturation)

• Controls and comparisons:

  • Known immunomodulatory proteins as benchmarks

  • Whole bacteria vs. purified protein comparisons

  • Strain-specific variations analysis

L. johnsonii has demonstrated immunomodulatory properties, altering macrophage and T-cell responses and regulating dendritic cell function , providing context for studying specific protein contributions to these effects.

How might emerging technologies advance understanding of LJ_1195 function?

Several cutting-edge technologies could accelerate functional characterization:

• CRISPR-Cas technologies:

  • CRISPRi for controlled gene expression modulation

  • Base editors for precise genomic modifications

  • CRISPR screening approaches to identify genetic interactions

• Single-cell technologies:

  • Single-cell RNA-Seq to capture population heterogeneity

  • Spatial transcriptomics to map host-microbe interactions

  • Mass cytometry for multiparameter cellular response analysis

• Advanced imaging approaches:

  • Super-resolution microscopy for protein localization

  • Live cell imaging to track dynamic processes

  • Correlative light and electron microscopy for structural-functional integration

• Computational advances:

  • Deep learning for protein function prediction

  • AlphaFold and similar AI tools for structure prediction

  • Advanced metabolic modeling incorporating protein-specific functions

These technologies could help resolve the currently unknown functions of the UPF0398 protein family and place LJ_1195 in specific metabolic and signaling pathways.

What research questions remain unresolved regarding LJ_1195 and related proteins?

Several critical questions warrant further investigation:

• Fundamental questions:

  • What is the precise biochemical function of LJ_1195?

  • How conserved is this function across Lactobacillus species?

  • What protein modifications are essential for its activity?

• Context-specific questions:

  • How does LJ_1195 expression vary across gut microenvironments?

  • What host factors influence its expression and activity?

  • Does LJ_1195 contribute to colonization efficiency?

• Translational questions:

  • Could recombinant LJ_1195 serve as a biomarker or therapeutic?

  • How might LJ_1195 contribute to probiotic effects?

  • Is LJ_1195 involved in bacterium-host communication pathways?

Addressing these questions would significantly advance understanding of both basic Lactobacillus biology and potential applications in microbiome modulation and probiotic development.