Recombinant Lactobacillus plantarum UPF0337 protein lp_1708 (lp_1708)

What are the optimal expression vectors for recombinant LP_1708 production in L. plantarum?

For recombinant protein expression in L. plantarum, several vector systems have demonstrated efficacy with comparable proteins. The pSIP series vectors (particularly pSIP409) offer inducible expression control via the sakacin promoter system, providing tight regulation of LP_1708 expression. These vectors typically incorporate signal peptides such as the Usp45 from Lactococcus lactis to facilitate protein secretion . Alternative vectors include pLp_1708FnBPA, which has been utilized for surface display applications in L. plantarum through fusion protein techniques similar to those employed with fibronectin binding proteins . For intracellular expression, pTRKH2 derivatives have shown success with recombinant proteins of similar molecular weight to LP_1708.

How can I verify successful transformation and expression of recombinant LP_1708 in L. plantarum?

Verification requires a multi-step approach. Begin with colony PCR utilizing LP_1708-specific primers similar to the recA-based primer design (LPrecAF and LPrecAR) used for L. plantarum identification . For protein expression confirmation, Western blot analysis using anti-His tag antibodies (if a His-tag was incorporated) represents the gold standard. Additionally, quantitative reverse transcription PCR (qRT-PCR) can verify transcription levels using the same methodology demonstrated effective for L. plantarum gene expression analysis . Flow cytometry can confirm surface expression if LP_1708 was engineered as a surface-displayed protein. Enzyme-linked immunosorbent assay (ELISA) provides quantitative assessment of protein yield, particularly for secreted constructs. Finally, functional activity assays specific to UPF0337 protein family characteristics should be developed to confirm biological activity.

What growth conditions optimize recombinant LP_1708 expression in L. plantarum cultures?

Optimal expression requires careful culture condition management. L. plantarum demonstrates highest growth and protein expression yields in MRS medium at 37°C under anaerobic conditions . Expression induction timing critically affects yield – initiate induction during early to mid-logarithmic phase (OD600 between 0.3-0.5) rather than later growth phases. Induction agent concentration requires optimization; for sakacin-inducible systems, 25-50 ng/ml of inducing peptide typically provides optimal expression while minimizing cellular stress. Temperature reduction to 30°C post-induction can enhance proper protein folding and reduce inclusion body formation. Additionally, supplementation with 2% glucose generally improves L. plantarum growth, though excessive glucose can repress certain promoters through carbon catabolite repression mechanisms .

What experimental approaches can determine the subcellular localization of LP_1708 in L. plantarum?

Multiple complementary approaches should be employed to definitively establish LP_1708 localization. Fractionation studies represent the foundation - separate cytoplasmic, membrane, and cell wall fractions using established L. plantarum fractionation protocols, followed by Western blot analysis of each fraction. Immunofluorescence microscopy using anti-LP_1708 antibodies with confocal imaging provides visual confirmation of localization patterns. For comprehensive analysis, cryo-electron microscopy can visualize protein distribution at higher resolution. Flow cytometry with labeled antibodies against LP_1708 quantitatively assesses surface expression if applicable. For dynamic localization studies, fluorescent protein fusions (such as GFP-LP_1708) enable real-time visualization, though validation is necessary to ensure fusion doesn't disrupt native localization. Predicted localization based on bioinformatic analysis of signal sequences and transmembrane domains provides initial hypotheses, but experimental verification remains essential .

How can I assess the impact of LP_1708 knockout or overexpression on L. plantarum immunomodulatory properties?

Immunomodulatory assessment requires both in vitro and in vivo experimental systems. For in vitro analysis, co-culture L. plantarum wild-type, LP_1708 knockout, and LP_1708 overexpression strains with bone marrow-derived dendritic cells (BMDCs) and measure dendritic cell differentiation and maturation markers (CD80, CD86, MHC-II) via flow cytometry . Quantify cytokine production profiles (particularly IL-6, IL-4, IL-17A) via ELISA and examine changes in cytokine mRNA levels through qRT-PCR . For T-cell stimulation assessment, co-culture modified L. plantarum strains with naive T-cells and measure T helper cell differentiation patterns, specifically examining shifts in Th1/Th2/Th17/Treg balances.

In vivo studies should examine mucosal immune responses following oral administration of modified strains. Analyze dendritic cell populations in Peyer's patches through flow cytometry, focusing on CD103+ and CD11c+ DC subsets . Measure B220+ B-cell production in mesenteric lymph nodes and Peyer's patches, and quantify LP_1708-specific IgG and secretory IgA (sIgA) antibody levels in serum and intestinal lavage samples, respectively . Additionally, evaluate changes in gut microbiota composition through 16S rDNA sequencing to detect indirect immunomodulatory effects resulting from LP_1708 modification .

What structural characterization methods are most appropriate for recombinant LP_1708?

A comprehensive structural characterization of LP_1708 requires multiple analytical techniques. X-ray crystallography provides the highest resolution structural information but requires protein crystallization optimization. Begin with high-purity protein isolated through affinity chromatography followed by size-exclusion chromatography to ensure monodispersity. Initial crystallization trials should test multiple buffer conditions (pH 4.5-9.0) and precipitants.

For solution-state analysis, circular dichroism spectroscopy determines secondary structure composition and thermal stability characteristics. Nuclear magnetic resonance (NMR) spectroscopy represents an alternative for full structure determination if crystallization proves challenging, though this typically requires isotopically labeled protein production. Small-angle X-ray scattering (SAXS) provides lower resolution structural information and insights into quaternary structure and protein flexibility.

Homology modeling based on structurally characterized UPF0337 family proteins provides initial structural hypotheses that can guide experimental designs. Differential scanning calorimetry measures thermodynamic stability parameters, while hydrogen-deuterium exchange mass spectrometry identifies surface-exposed regions and potential interaction interfaces. For glycosylation characterization, mass spectrometry analysis following glycosidase treatments can identify and map post-translational modifications that may affect LP_1708 function .

What are the most effective transformation methods for introducing LP_1708 constructs into L. plantarum?

Electroporation remains the most reliable transformation method for L. plantarum. Optimal results require preparing electrocompetent cells from mid-logarithmic phase cultures (OD600 of 0.4-0.6) grown in MRS medium with 1% glycine to weaken cell walls. After harvesting, cells should undergo multiple washing steps in decreasing concentrations of sucrose buffer (final concentration: 0.3M sucrose). For electroporation, parameters of 2.0 kV, 25 μF, and 200 Ω typically yield highest transformation efficiencies. Critical factors affecting transformation success include DNA purity (use commercial kits that remove protein contamination), plasmid size (smaller constructs transform more efficiently), and methylation status (L. plantarum contains restriction systems that may degrade inappropriately methylated DNA) .

Alternative approaches include conjugation from Escherichia coli donors, which can be advantageous for larger constructs but requires donor strains carrying mobilization functions. Protoplast transformation represents another option, though it demonstrates lower efficiency and requires optimized regeneration conditions. For genomic integration of LP_1708 constructs, CRISPR-Cas9 systems have been adapted for L. plantarum, offering precise genomic modifications through homology-directed repair mechanisms following targeted DNA cleavage .

How can I develop a multiplex PCR method to simultaneously detect LP_1708 and track L. plantarum colonization in vivo?

A robust multiplex PCR system requires carefully designed primer and probe sets with similar amplification efficiencies but distinct target specificities. For LP_1708 detection, design primers targeting unique regions of the LP_1708 gene with amplicon size between 100-200 bp for optimal qPCR performance. For L. plantarum identification, the recA gene provides a reliable target with established primer sets (LPrecAF/LPrecAR) .

To enable multiplex discrimination, develop TaqMan probes with different fluorophores - 6-carboxyfluorescein (FAM) for LP_1708 and 2′-chloro-7′-phenyl-4-dichloro-6-carboxyfluorescein (VIC) for the L. plantarum recA gene, each with minor groove binder (MGB) quenchers . Validate the multiplex system using DNA from pure cultures with concentrations ranging from 10^1 to 10^9 cells/ml to establish detection limits and standard curves.

For in vivo tracking, extract DNA from fecal or tissue samples using commercial kits designed for PCR inhibitor removal. Normalize quantification using spike-in controls such as Bacillus thuringiensis with its species-specific plcR gene target . PCR conditions should include initial denaturation at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute, with appropriate positive and negative controls included in each run .

What strategies can overcome codon usage bias when expressing LP_1708 in different host systems?

Codon optimization represents a critical consideration when expressing LP_1708 in heterologous hosts. For expression in L. plantarum, analyze the native LP_1708 sequence for rare codons that might limit translation efficiency. Software tools like OPTIMIZER or JCat can generate optimized sequences based on L. plantarum codon usage tables. Key optimization parameters include Codon Adaptation Index (CAI) improvement, GC content normalization (aim for 45-50% in L. plantarum), and elimination of mRNA secondary structures near the ribosome binding site.

For expression in E. coli as a preliminary research system, more extensive optimization is necessary due to greater phylogenetic distance. Consider using E. coli strains engineered to supply rare tRNAs (such as Rosetta or CodonPlus strains) if full codon optimization is not feasible. For mammalian cell expression, remove prokaryotic regulatory elements and optimize for mammalian codon preferences.

Additionally, inclusion of an N-terminal fusion partner like thioredoxin or SUMO can enhance solubility and expression levels of heterologous proteins in various host systems. Finally, synthetic gene synthesis offers the most straightforward implementation of codon optimization, allowing simultaneous introduction of useful restriction sites while eliminating problematic secondary structures .

How can I determine if LP_1708 affects L. plantarum adherence to intestinal epithelial cells?

Adherence assessment requires multiple complementary in vitro and ex vivo approaches. Begin with cell culture models using intestinal epithelial cell lines such as Caco-2, HT-29, or the porcine IPEC-J2 line which has demonstrated utility in L. plantarum adhesion studies . Prepare wild-type, LP_1708 knockout, and LP_1708 overexpression L. plantarum strains standardized to equivalent CFU/ml (typically 10^8 CFU/ml). Following co-incubation for 1-2 hours, quantify adherent bacteria through washing steps followed by either plate counting or qPCR quantification.

Fluorescent labeling of bacterial strains (using CFDA-SE or similar vital dyes) enables flow cytometric quantification of adherence and provides higher throughput than plating methods. Confocal microscopy with fluorescently labeled bacteria offers visual confirmation and spatial distribution analysis of adherence patterns. For higher physiological relevance, ex vivo adherence assays using freshly isolated intestinal tissue sections can be performed, though these require more complex methodology and animal ethics approval.

To elucidate mechanisms, examine whether LP_1708 affects surface properties through hydrophobicity assays (microbial adhesion to hydrocarbons), aggregation tests, and biofilm formation capacity. Additionally, test whether recombinant purified LP_1708 protein can competitively inhibit bacterial adhesion, which would suggest direct interaction with epithelial receptors .

What proteomics approaches can identify potential interaction partners of LP_1708?

Multiple complementary proteomics strategies can identify LP_1708 interaction partners. Begin with affinity purification coupled with mass spectrometry (AP-MS) using either epitope-tagged LP_1708 (His, FLAG, or HA tag) or antibodies against native LP_1708. Following stringently controlled pull-down experiments, identify co-precipitating proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Implement SILAC (Stable Isotope Labeling with Amino acids in Cell culture) methodology to distinguish true interactors from background contaminants.

For in situ detection of interactions, proximity labeling approaches using BioID or APEX2 fused to LP_1708 enable biotinylation of proximal proteins, which can then be isolated through streptavidin affinity purification and identified by MS. Crosslinking mass spectrometry (XL-MS) provides additional structural information by chemically linking interaction interfaces before MS analysis.

Two-hybrid systems (bacterial or yeast) offer complementary genetic approaches to screen for binary interactions, though these require library construction from L. plantarum genomic DNA. For confirmation of specific interactions, surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) provides quantitative binding parameters between purified LP_1708 and candidate interactors.

Computational predictions using tools like STRING database can guide experimental designs by identifying high-probability interaction candidates based on genomic context, co-expression patterns, and evolutionary conservation .

How can transcriptomic analysis elucidate the role of LP_1708 in L. plantarum physiology?

RNA sequencing (RNA-Seq) provides the most comprehensive approach to analyze transcriptome-wide changes resulting from LP_1708 modification. Compare wild-type L. plantarum with LP_1708 knockout and overexpression strains under standardized growth conditions. For physiologically relevant insights, include experimental conditions that might trigger LP_1708 function, such as acid stress, bile exposure, nutrient limitation, or host cell contact.

Sample collection timing is critical - harvest RNA at multiple growth phases (early logarithmic, mid-logarithmic, and stationary) to capture temporal dynamics. Employ stringent RNA extraction protocols optimized for gram-positive bacteria, which typically require enzymatic cell wall digestion followed by TRIzol extraction. Validate differential expression of key genes via quantitative RT-PCR using methodology similar to that employed for IL-6 gene expression analysis in L. plantarum studies .

For data analysis, identify differentially expressed genes and perform functional enrichment analysis to determine biological processes, molecular functions, and cellular components affected by LP_1708 status. Construction of gene regulatory networks can reveal master regulators potentially mediating LP_1708 effects. Integration with metabolomics data provides deeper insights into metabolic pathway alterations.

Consider ChIP-Seq (Chromatin Immunoprecipitation Sequencing) if LP_1708 is hypothesized to function as a transcription factor or DNA-binding protein, which would identify direct genomic binding sites .

How does LP_1708 potentially influence L. plantarum's ability to modulate epithelial barrier function?

LP_1708's impact on epithelial barrier function can be assessed through transepithelial electrical resistance (TEER) measurements using polarized intestinal epithelial cell monolayers grown on Transwell inserts. Compare TEER changes when monolayers are exposed to wild-type, LP_1708 knockout, and LP_1708 overexpression L. plantarum strains. Complement TEER measurements with paracellular permeability assays using fluorescent markers such as FITC-dextran or Lucifer yellow.

At the molecular level, analyze tight junction protein expression and localization through Western blotting and immunofluorescence microscopy, focusing on zonula occludens-1 (ZO-1), occludin, and claudins. These proteins have demonstrated responsiveness to L. plantarum exposure in previous studies . Examine whether LP_1708 influences TLR-2 signaling pathways, as L. plantarum has been shown to enhance tight junction integrity through TLR-2 activation .

For mechanistic insights, investigate whether LP_1708 affects L. plantarum's production of metabolites known to influence barrier function, such as short-chain fatty acids. Additionally, measure epithelial cytokine responses, particularly IL-6, which has been associated with L. plantarum-mediated immunomodulation .

In vivo validation could employ intestinal permeability assays in mice following oral administration of the different bacterial strains, measuring translocation of FITC-dextran from the intestinal lumen to the bloodstream .

What methodologies can determine if LP_1708 contributes to L. plantarum persistence in the gastrointestinal tract?

Persistence assessment requires combining in vivo colonization studies with molecular tracking techniques. Design feeding studies where germ-free or conventional mice receive single or multiple doses of wild-type, LP_1708 knockout, and LP_1708 overexpression L. plantarum strains. For quantitative tracking, develop strain-specific primers for qPCR detection in fecal samples, similar to the established recA-based detection systems for L. plantarum .

Sample collection timing is critical - collect fecal samples at baseline, during administration, and for extended periods post-administration (up to 6 months) to detect potential long-term colonization differences . For spatial distribution analysis, perform quantitative culture and qPCR on intestinal contents and mucosal scrapings from different gastrointestinal compartments following sacrifice at defined timepoints.

Competitive index experiments provide higher sensitivity - administer equal mixtures of wild-type and LP_1708 mutant strains and quantify their relative abundance over time, which can reveal subtle fitness differences. For mechanistic insights, measure parameters potentially affecting persistence: acid tolerance, bile resistance, adhesion to mucus (using mucin-coated plates), and biofilm formation capacity.

Consider metagenomic sequencing of the intestinal microbiota to detect whether LP_1708 status affects interactions with other microbiota members, potentially indirectly influencing persistence .

How can I evaluate whether LP_1708 affects L. plantarum's immunomodulatory properties in different disease models?

Disease model selection should target conditions where L. plantarum has demonstrated therapeutic potential. For inflammatory bowel disease models, employ dextran sodium sulfate (DSS)-induced colitis or IL-10 knockout mice. Administer wild-type, LP_1708 knockout, and LP_1708 overexpression L. plantarum strains prophylactically or therapeutically and assess clinical parameters (weight loss, stool consistency, bleeding) and histopathological scores.

For metabolic disease models, utilize high-fat diet-induced obesity models and measure metabolic parameters including glucose tolerance, insulin resistance, and adipose tissue inflammation following administration of the different bacterial strains. In allergy models, evaluate the capacity of modified L. plantarum strains to prevent sensitization to model allergens and subsequent inflammatory responses.

Across all models, comprehensive immunophenotyping is essential - analyze dendritic cell populations with a focus on tolerogenic DC markers, T cell subsets with particular attention to Th1/Th2/Th17/Treg balances, and B cell responses including antibody production profiles . These analyses should be performed in relevant tissues including Peyer's patches, mesenteric lymph nodes, and the spleen.

Cytokine profiling through multiplex assays should examine both pro-inflammatory (TNF-α, IL-6, IL-1β) and regulatory cytokines (IL-10, TGF-β). Gene expression analysis of key immune mediators in intestinal tissue using qPCR or RNA-Seq provides additional mechanistic insights .