Recombinant Lactobacillus johnsonii UPF0337 protein LJ_0034.1 (LJ_0034.1)

What is the molecular structure of Lactobacillus johnsonii UPF0337 protein LJ_0034.1?

The UPF0337 protein LJ_0034.1 from L. johnsonii belongs to an uncharacterized protein family. While specific structural data for this protein is still emerging, researchers typically approach structural characterization through a combination of computational prediction and experimental methods. X-ray crystallography or NMR spectroscopy should be considered for definitive structural determination. When working with recombinant forms, proteins are typically expressed with tags (such as His-tags) to facilitate purification, and careful consideration of tag position is necessary to avoid interference with protein folding and function .

What expression systems are optimal for producing recombinant L. johnsonii proteins?

The growth medium composition particularly affects yield, as demonstrated in studies with other L. johnsonii strains where optimized media increased viable cell counts significantly compared to standard media formulations .

How should recombinant L. johnsonii proteins be stored to maintain stability?

Recombinant proteins derived from L. johnsonii should be stored following protocols similar to other recombinant proteins. Lyophilization from a filtered solution in PBS is recommended for long-term storage. Upon reconstitution (typically at 100 μg/mL in sterile PBS), aliquot the protein to avoid repeated freeze-thaw cycles. Storage in a manual defrost freezer at -20°C (for short-term) or -80°C (for long-term) is advisable. For proteins without carrier proteins (carrier-free), stability may be compromised, necessitating more careful handling .

What approaches are most effective for characterizing the function of uncharacterized proteins like UPF0337 protein LJ_0034.1?

When working with uncharacterized proteins such as UPF0337 protein LJ_0034.1, a multi-faceted approach is recommended:

• Computational analysis: Use bioinformatics tools to predict function based on sequence homology, domain architecture, and phylogenetic relationships

• Genetic context analysis: Examine neighboring genes in the L. johnsonii genome for functional clues

• Protein-protein interaction studies: Employ pull-down assays, co-immunoprecipitation, or yeast two-hybrid screening

• Knockout/knockdown studies: Generate L. johnsonii strains lacking the protein and observe phenotypic changes

• Heterologous expression: Express the protein in model organisms to observe gain-of-function effects

Given that L. johnsonii demonstrates bile salt hydrolase (BSH) activity that contributes to its probiotic effects, investigating whether UPF0337 protein LJ_0034.1 interacts with or modulates this pathway could be particularly informative .

How can researchers optimize purification protocols for L. johnsonii recombinant proteins?

Purification of recombinant L. johnsonii proteins requires systematic optimization. A stepwise approach includes:

• Expression optimization: Adjust conditions to maximize soluble protein expression

• Lysis buffer optimization: Test different pH conditions and detergent concentrations

• Purification strategy: For His-tagged constructs, optimize imidazole concentrations in binding and elution buffers

• Quality control: Verify purity using SDS-PAGE and Western blotting

• Functionality assessment: Confirm that the purified protein retains biological activity

For recombinant L. johnsonii proteins, which may have specific stability requirements, incorporating protease inhibitors and maintaining cold temperatures throughout purification is especially important. Additionally, if the protein demonstrates autolytic properties, which has been observed in some L. johnsonii derivatives, consider testing various buffer compositions to minimize degradation during purification .

What controls should be included when evaluating recombinant L. johnsonii protein function in cell culture?

When assessing the function of recombinant L. johnsonii proteins in cell culture systems, the following controls are essential:

• Negative control: Buffer-only treatment to establish baseline cellular responses

• Vehicle control: Carrier protein (e.g., BSA) at equivalent concentrations

• Heat-inactivated protein: To distinguish between effects of protein structure versus sequence

• Dose-response curve: Multiple concentrations to establish biological relevance

• Time-course analysis: Multiple time points to characterize the kinetics of response

For adhesion studies specifically, which are relevant for L. johnsonii proteins that may mediate bacterial interactions with host cells, including established reference strains such as Lacticaseibacillus rhamnosus GG (LGG) provides important comparative data .

How does the UPF0337 protein LJ_0034.1 potentially contribute to L. johnsonii's probiotic functions?

Understanding the contribution of UPF0337 protein LJ_0034.1 to L. johnsonii's probiotic properties requires integration of genetic, biochemical, and physiological approaches. L. johnsonii demonstrates several probiotic mechanisms, including:

• Modulation of host immune responses (reduction of IL-4, IL-5, IL-13, and IL-17)

• Production of antimicrobial compounds against pathogens

• Competition with pathogens for epithelial adhesion sites

• Bile salt hydrolase (BSH) activity that converts conjugated bile acids to free forms

To investigate UPF0337 protein LJ_0034.1's role in these functions, researchers should consider:

• Creating knockout strains lacking the gene encoding UPF0337 protein LJ_0034.1

• Comparing wild-type and knockout strains in functional assays measuring adhesion to intestinal cell lines

• Assessing biofilm formation capabilities, which relate to colonization potential

• Measuring BSH activity in the presence and absence of the protein

• Evaluating stress resistance, particularly to bile acids and low pH environments

What is the relationship between protein structure and function for UPF0337 protein family members?

The UPF0337 protein family remains largely uncharacterized, making structure-function relationships particularly valuable to establish. Research approaches should include:

• Comparative structural analysis across UPF0337 family members from different bacterial species

• Identification of conserved residues that may indicate functional importance

• Site-directed mutagenesis of these conserved residues followed by functional assays

• Construction of chimeric proteins with domains from different UPF0337 family members

• Crystallization trials with and without potential binding partners

When analyzing structural features, researchers should pay particular attention to regions that might mediate protein-protein interactions or enzymatic activity, as these could provide insights into the biological role of UPF0337 protein LJ_0034.1 in L. johnsonii.

How can adaptive laboratory evolution be applied to enhance specific properties of L. johnsonii strains expressing modified UPF0337 proteins?

Adaptive laboratory evolution (ALE) offers a powerful approach to developing enhanced L. johnsonii strains. This methodology has successfully generated stress-resistant derivatives of L. johnsonii with improved probiotic properties . For researchers interested in UPF0337 protein modification, the following strategy could be employed:

• Design selective pressures relevant to UPF0337 protein function

• Implement sequential screening approaches, such as:

  • Growth under increasing concentrations of relevant stressors

  • Selection for modified surface properties or adhesion capabilities

  • Screening for enhanced stability under gastrointestinal conditions

This approach has yielded derivatives with significantly improved properties, including enhanced resistance to bile acids, reduced autolysis, and increased adhesion to intestinal cell lines as demonstrated in previous L. johnsonii studies .

What analytical methods should be used to assess the purity and integrity of recombinant UPF0337 protein LJ_0034.1?

Multiple analytical techniques should be employed to comprehensively characterize recombinant UPF0337 protein LJ_0034.1:

• SDS-PAGE: For assessment of purity and approximate molecular weight

• Western blotting: For specific identification using antibodies against the protein or epitope tags

• Size-exclusion chromatography: To evaluate oligomerization state and aggregation propensity

• Mass spectrometry: For precise molecular weight determination and post-translational modification analysis

• Circular dichroism: To assess secondary structure composition

• Thermal shift assay: To evaluate protein stability under various buffer conditions

When analyzing recombinant proteins from L. johnsonii, researchers should be particularly attentive to potential proteolytic degradation, as these bacteria produce various proteases that could co-purify with the target protein .

How can researchers troubleshoot expression and solubility issues with recombinant L. johnsonii proteins?

Recombinant proteins from L. johnsonii may present expression and solubility challenges. A systematic troubleshooting approach includes:

• Expression vector optimization:

  • Test different promoter strengths

  • Try various fusion tags (His, GST, MBP) in both N- and C-terminal positions

  • Consider codon optimization for the expression host

• Expression conditions adjustment:

  • Reduce induction temperature (16-20°C)

  • Lower inducer concentration

  • Extend expression time

• Solubility enhancement strategies:

  • Add solubility-enhancing compounds to lysis buffer (glycerol, mild detergents)

  • Co-express with molecular chaperones

  • Consider in vitro refolding if inclusion bodies form

• Alternative expression systems:

  • If E. coli proves unsuitable, consider Lactobacillus expression systems or insect cell systems

For L. johnsonii proteins specifically, selecting optimal growth media components can significantly impact yield, with combinations of soybean meal, molasses, and sodium acetate showing particular promise .

What are the challenges in differentiating the functions of native versus recombinant forms of L. johnsonii proteins?

Researchers face several challenges when comparing native versus recombinant L. johnsonii proteins:

• Post-translational modifications: L. johnsonii may modify proteins in ways that heterologous expression systems cannot replicate

• Protein-protein interactions: Native proteins exist in a complex cellular milieu that affects their function

• Conformational differences: Recombinant tags or expression conditions may alter protein folding

• Activity assessment: Developing assays that accurately measure physiologically relevant activities

To address these challenges, researchers should:

• Compare the biochemical properties of native (extracted from L. johnsonii) and recombinant proteins

• Assess whether recombinant proteins can complement knockout strains

• Use structure-guided approaches to design recombinant proteins minimally affected by tags

• Consider expressing proteins in closely related Lactobacillus species rather than E. coli

L. johnsonii proteins associated with probiotic functions, such as those involved in adhesion or immunomodulation, are particularly susceptible to functional differences between native and recombinant forms .

How might UPF0337 protein LJ_0034.1 contribute to L. johnsonii's immunomodulatory effects?

L. johnsonii exerts significant immunomodulatory effects, including downregulation of pro-inflammatory cytokines (IL-4, IL-5, IL-13, IL-17, IL-6, IL-1β, TNFα) and upregulation of beneficial factors like IFNβ and IFN-γ . To investigate whether UPF0337 protein LJ_0034.1 contributes to these effects, researchers could:

• Compare cytokine profiles induced by wild-type L. johnsonii versus strains with altered UPF0337 protein LJ_0034.1 expression

• Test purified recombinant UPF0337 protein directly on immune cells and measure cytokine responses

• Investigate potential structural similarities between UPF0337 protein and known immunomodulatory molecules

• Assess whether UPF0337 protein interacts with pattern recognition receptors on immune cells

• Evaluate UPF0337 protein's role in L. johnsonii's protective effects against respiratory syncytial virus (RSV) and allergic airway diseases

This research direction is particularly relevant given L. johnsonii's demonstrated capacity to modulate immune responses in various disease models .

What strategies can optimize the stability of recombinant UPF0337 protein for research applications?

Enhancing the stability of recombinant UPF0337 protein requires consideration of multiple factors:

• Buffer optimization:

  • Screen various pH conditions (typically 6.5-8.0)

  • Test stabilizing additives (glycerol, trehalose, arginine)

  • Include appropriate reducing agents if the protein contains cysteines

• Storage formulation:

  • Lyophilize from a 0.2 μm filtered solution in PBS

  • Consider carrier proteins (e.g., BSA) to prevent surface adsorption and aggregation

  • Store at -80°C in small aliquots to prevent freeze-thaw damage

• Protein engineering approaches:

  • Identify and mutate surface residues prone to oxidation or degradation

  • Consider disulfide engineering to enhance conformational stability

  • Remove protease-sensitive regions if not essential for function

For L. johnsonii proteins specifically, derivatives with reduced autolysis have been developed through adaptive evolution, suggesting that certain modifications can enhance stability without compromising function .

How can researchers assess the potential interaction between UPF0337 protein LJ_0034.1 and host intestinal cells?

Investigating interactions between UPF0337 protein and intestinal cells requires a multi-faceted approach:

• Adhesion assays:

  • Test binding of labeled recombinant UPF0337 protein to intestinal cell lines (Caco-2, HT-29, HT-29 MTX, T84)

  • Compare adhesion of wild-type L. johnsonii versus strains with altered UPF0337 protein expression

  • Use confocal microscopy to visualize protein localization on cell surfaces

• Receptor identification:

  • Conduct pull-down experiments using UPF0337 protein as bait with intestinal cell lysates

  • Perform cross-linking studies followed by mass spectrometry to identify binding partners

  • Use surface plasmon resonance to measure binding kinetics with candidate receptors

• Functional consequences:

  • Measure changes in barrier function (transepithelial electrical resistance)

  • Assess modulation of mucin production in goblet cell models

  • Evaluate effects on intestinal cell cytokine production

Previous studies with L. johnsonii have demonstrated significant adhesion to intestinal cell lines with strain-specific variations, suggesting that particular proteins contribute significantly to this property .