Recombinant Lactobacillus plantarum UPF0291 protein lp_2062 (lp_2062)

What is Recombinant Lactobacillus plantarum UPF0291 protein lp_2062?

Recombinant UPF0291 protein lp_2062 is a protein that can be expressed in various host systems, including bacterial, yeast, insect, and mammalian cells. This protein, derived from Lactobacillus plantarum, is part of the UPF0291 protein family and is encoded by the lp_2062 gene. The recombinant form refers to the protein expressed through genetic engineering techniques, where the gene encoding lp_2062 is inserted into expression vectors and transformed into appropriate host cells for protein production .

Which expression systems are optimal for producing recombinant UPF0291 protein lp_2062?

Multiple expression systems can be utilized for the production of recombinant UPF0291 protein lp_2062, each with distinct advantages:

The selection of an expression system should be based on the specific research requirements, including desired protein yield, post-translational modifications needed for correct protein folding, and the intended application of the purified protein .

What key factors influence successful expression of recombinant proteins in L. plantarum?

Several critical factors determine the success of recombinant protein expression in L. plantarum:

How can researchers optimize codon usage for enhanced expression of UPF0291 protein lp_2062?

Codon optimization represents a critical step in maximizing recombinant protein expression in L. plantarum. The process involves several key methodological steps:

• Analysis of codon usage bias: Researchers should first analyze the codon usage patterns in highly expressed L. plantarum genes to identify preferred codons.

• Gene synthesis approach: The lp_2062 gene should be synthesized with optimized codons according to L. plantarum's codon usage bias. Commercial services (e.g., GenScript) can synthesize these optimized gene fragments .

• Vector cloning strategy: The optimized gene sequence should be cloned into appropriate expression vectors, such as derivatives of the pSIP401 vector series, which are specifically designed for inducible gene expression in Lactobacillus species .

• Verification of expression efficiency: Comparative analysis between optimized and non-optimized sequences should be performed to quantify improvement in expression levels, typically using Western blotting or fluorescence-based quantification methods .

Studies have shown that codon optimization can significantly enhance the expression levels of recombinant proteins in L. plantarum, with some reports indicating several-fold increases in protein yield .

What are the established protocols for constructing recombinant L. plantarum strains expressing UPF0291 protein lp_2062?

Construction of recombinant L. plantarum strains expressing UPF0291 protein lp_2062 involves a systematic approach:

• Gene preparation: Synthesize the lp_2062 gene with optimized codons for L. plantarum expression .

• Signal peptide and anchor selection: For surface display, link endogenous signal peptides (e.g., 1320/ALX04_001320) to the 5′ terminus of the optimized gene. For anchoring, add appropriate anchor sequences such as the cell wall anchor sequence comprising C-terminal residues from Lp_2578 (including the LPQTSE motif) .

• Vector construction: Clone the prepared gene construct into an appropriate expression vector:

  • Amplify the gene using specific primers with restriction enzyme sites

  • Digest the vector (e.g., pSIP401) with appropriate restriction enzymes (e.g., NcoI/HindIII)

  • Ligate the gene into the digested vector using Gibson Assembly or similar techniques

• Transformation: Transform the expression plasmid into competent L. plantarum cells using electroporation, with selection on media containing appropriate antibiotics (e.g., 5 μg/mL erythromycin for erm-based systems) .

• Expression verification: Verify successful expression through methods such as:

  • SDS-PAGE analysis

  • Western blotting

  • Immunofluorescence staining with FITC-labeled antibodies

  • Transmission electron microscopy for visualizing surface-displayed proteins

What analytical methods are most effective for characterizing the expression and stability of UPF0291 protein lp_2062?

Several analytical methods can be employed to comprehensively characterize the expression and stability of recombinant UPF0291 protein lp_2062:

• Expression level analysis:

  • SDS-PAGE for basic protein visualization

  • Western blotting for specific detection using appropriate antibodies

  • Quantitative ELISA for precise concentration determination

• Cellular localization:

  • Cell fractionation to identify protein distribution between cell wall, membrane, and cytoplasmic fractions

  • Immunofluorescence microscopy using labeled antibodies to visualize surface expression

  • Transmission electron microscopy for high-resolution localization studies

• Stability assessment:

  • Temperature stability testing (e.g., at normal conditions and elevated temperatures such as 50°C)

  • pH stability evaluation (e.g., at physiological pH and acidic conditions such as pH 1.5)

  • Salt concentration tolerance testing

  • Long-term storage stability analysis

• Functional characterization:

  • Binding assays to evaluate interaction with target molecules

  • Activity assays specific to the protein's function

  • Antigenicity testing if the protein has immunological applications

The combination of these methods provides a comprehensive profile of the recombinant protein's expression characteristics and stability under various conditions relevant to its intended applications .

How can recombinant L. plantarum expressing UPF0291 protein lp_2062 be optimized for mucosal vaccine delivery?

Optimizing recombinant L. plantarum for mucosal vaccine delivery involves several advanced strategies:

• Surface display engineering: Utilize cell wall anchor sequences (e.g., from Lp_2578) to efficiently display lp_2062 on the bacterial surface, enhancing direct interaction with mucosal immune cells. The anchor typically consists of a linker region followed by an LPQTSE motif, a hydrophobic stretch, and a positively charged C-terminal .

• Targeting enhancement: Fuse the lp_2062 protein with specific targeting peptides to enhance uptake by mucosal immune cells:

  • Dendritic cell (DC)-targeting peptides (e.g., DCpep)

  • M cell-targeting peptides to enhance transport across the intestinal epithelium

• Immunization protocol optimization: Develop optimized administration protocols:

  • For oral immunization: typically 10^9 CFU/100 μL administered in prime-boost-boost regimens (e.g., days 1-3, 14-16, and 29-31)

  • For intranasal immunization: 10^7 cells in 10 μL (or 1 μL containing 10^7 cells administered directly to nostrils under anesthesia)

• Stability enhancement: Ensure the recombinant protein maintains stability under GIT conditions:

  • Confirm strain survival at low pH (e.g., pH 3) and in the presence of bile salts (0.3%)

  • Evaluate adhesion properties to intestinal epithelial cells using in vitro models such as Caco-2 cell lines

• Immune response monitoring: Assess the induction of mucosal, cellular, and systemic immune responses through appropriate sample collection and analysis from various tissues (intestinal mucosa, respiratory airways) .

These approaches have been successfully implemented for other recombinant proteins in L. plantarum and could be adapted for UPF0291 protein lp_2062 applications .

What in vivo imaging techniques can be employed to study the colonization dynamics of recombinant L. plantarum strains?

Advanced in vivo imaging techniques offer valuable insights into the colonization dynamics of recombinant L. plantarum strains:

• Fluorescent protein expression systems:

  • Co-expression of fluorescent proteins (e.g., mCherry or IRFP713) alongside the protein of interest

  • Construction of expression vectors (e.g., pSIP401-mcherry or pSIP401-IRFP713) for transformation into L. plantarum

• Real-time in vivo imaging:

  • Utilize multimodal imaging systems such as IVIS Lumina XR (PerkinElmer)

  • Remove background fluorescence using adaptive background subtraction tools

  • Analyze images with specialized software (e.g., Living Image version 4.3.1)

• Gastrointestinal transit studies:

  • Administer fluorescently labeled recombinant bacteria (typically 10^9 CFU/100 μL)

  • Harvest intestines at predetermined time points (2, 4, 6, 12, 24, 48, 72, and 96 hours post-administration)

  • Image the entire gastrointestinal tract to track bacterial movement

  • Segment and analyze specific intestinal regions (duodenum, jejunum, cecum, colon) separately

• Quantitative assessment of colonization:

  • Wash intestinal mucosa with PBS to collect attached bacteria

  • Quantify bacterial abundance using plate coating methods

  • Compare colonization patterns between different intestinal segments and at various time points

These imaging techniques provide valuable data on the persistence, transit time, and localization preferences of recombinant L. plantarum strains, which is essential for optimizing delivery strategies for UPF0291 protein lp_2062 .

How do different induction systems affect the expression kinetics of recombinant proteins in L. plantarum?

The choice and optimization of induction systems significantly impact expression kinetics of recombinant proteins in L. plantarum:

• Peptide pheromone induction systems:

  • SppIP induction: Optimal concentration typically around 50 ng/mL, with peak expression occurring between 6-10 hours post-induction at 37°C

  • IP-673 induction: Effective at 25 ng/mL when cultures reach an OD600 of 0.3, with expression typically evaluated 6 hours post-induction

• Expression kinetics parameters:

  Induction Parameter	Typical Range	Optimization Considerations
  Cell density at induction	OD600 0.3-0.5	Earlier induction may increase per-cell yield but decrease total biomass
  Inducer concentration	25-50 ng/mL	Higher concentrations may not improve yield and may increase costs
  Post-induction time	6-10 hours	Longer times may lead to protein degradation or cell lysis
  Induction temperature	30-37°C	Lower temperatures may improve folding of complex proteins

• Monitoring expression progression:

  • SDS-PAGE analysis of samples collected at various time points post-induction

  • Western blotting to quantify target protein accumulation over time

  • Immunofluorescence microscopy to visualize surface expression dynamics

• Harvest considerations:

  • Centrifugation at 5,000× g for 5-10 minutes to collect cells

  • PBS washing to remove media components that might interfere with downstream analysis

  • Appropriate storage conditions (-20°C) to preserve samples before analysis

Understanding and optimizing these induction parameters allows researchers to maximize the yield and quality of recombinant UPF0291 protein lp_2062 produced in L. plantarum expression systems .

What strategies address poor expression levels of UPF0291 protein lp_2062 in L. plantarum systems?

When facing challenges with low expression levels of UPF0291 protein lp_2062, researchers can implement several strategic approaches:

• Vector optimization:

  • Evaluate promoter strength and compatibility

  • Consider alternative modular vectors from the pSIP series

  • Ensure proper orientation and reading frame of the inserted gene

• Codon optimization refinement:

  • Re-analyze codon usage patterns specifically for highly expressed proteins in L. plantarum

  • Focus optimization on the N-terminal region (first 50-100 codons), which has the strongest impact on translation initiation

  • Eliminate rare codons that might cause ribosomal stalling

• Signal peptide and anchor modifications:

  • Test alternative signal peptides (e.g., switching from Lp_1261 to ALX04_001320)

  • Optimize the linker region between the protein and anchor domain

  • Ensure correct incorporation of targeting motifs (e.g., LPQTSE)

• Culture and induction condition optimization:

  • Systematically vary induction time points (OD600 values from 0.1 to 0.5)

  • Test inducer concentration ranges (10-100 ng/mL)

  • Evaluate temperature effects (30°C, 37°C, 42°C)

  • Modify media composition (e.g., carbon source, nitrogen availability)

• Protein stability enhancement:

  • Co-express chaperone proteins to aid folding

  • Include protease inhibitors during cell lysis

  • Evaluate protein half-life and implement strategies to reduce degradation

Implementing these strategies in a systematic manner, with appropriate controls at each step, allows for the identification and resolution of specific bottlenecks in the expression of UPF0291 protein lp_2062 .

How can researchers address stability challenges when working with UPF0291 protein lp_2062?

Stability challenges with UPF0291 protein lp_2062 can be addressed through methodical approaches targeting various stages of protein production and analysis:

• Expression-phase stability optimization:

  • Adjust growth temperature during expression (lower temperatures often improve folding and stability)

  • Optimize media composition to provide necessary cofactors or stabilizing agents

  • Consider co-expression of molecular chaperones to assist proper folding

• Extraction and purification considerations:

  • Incorporate stabilizing agents in lysis buffers (glycerol, specific ions, mild detergents)

  • Optimize purification protocols to minimize time and potentially destabilizing conditions

  • Consider affinity tags that allow rapid, gentle purification under native conditions

• Stability testing protocols:

  • Evaluate temperature stability across relevant ranges (4°C, 25°C, 37°C, 50°C)

  • Test pH stability, particularly at physiologically relevant conditions (pH 1.5-8.0)

  • Assess stability in the presence of varying salt concentrations

  • Determine storage stability under different conditions (solution vs. lyophilized, different buffer compositions)

• Formulation optimization:

  • Identify optimal buffer compositions through differential scanning fluorimetry

  • Test various additives (sugars, amino acids, polyols) for stabilizing effects

  • Evaluate protective excipients for freeze-thaw stability if cryopreservation is needed

• Analytical methods for stability monitoring:

  • Size-exclusion chromatography to detect aggregation

  • Circular dichroism to monitor secondary structure changes

  • Activity assays to correlate structural stability with functional retention

Previous studies with recombinant proteins in L. plantarum have demonstrated that properly expressed recombinant proteins can maintain stability under challenging conditions (50°C, pH 1.5, high salt), suggesting similar approaches may be effective for UPF0291 protein lp_2062 .

What experimental design approaches help optimize surface display efficiency of UPF0291 protein lp_2062?

Optimizing surface display efficiency of UPF0291 protein lp_2062 requires systematic experimental design approaches:

• Anchor domain optimization:

  • Comparative testing of different anchor domains:

    • Lp_2578 anchor (223 C-terminal residues, including 189-residue linker region and LPQTSE motif)

    • Alternative anchors with varying linker lengths

  • Evaluation of anchor position (N-terminal vs. C-terminal fusion)

• Fusion construct design strategies:

  • Incorporation of specific peptide tags:

    • DCpep for targeting dendritic cells

    • M cell-targeting peptides for enhanced mucosal uptake

  • Addition of spacer sequences between protein domains to reduce steric hindrance

  • Testing of multiple construct designs in parallel

• Expression verification methods:

  • Western blot analysis of cell wall fractions to confirm surface localization

  • Immunofluorescence microscopy using FITC-labeled secondary antibodies

  • Flow cytometry to quantify the percentage of cells displaying the protein

  • Transmission electron microscopy for high-resolution visualization of surface expression

• Factorial experimental design approach:

  Factor	Levels to Test	Measurement Method
  Signal peptide	Lp_1261, ALX04_001320, others	Western blot band intensity
  Anchor domain	Lp_2578, alternative anchors	Immunofluorescence intensity
  Linker length	Short (50 aa), Medium (100 aa), Long (189 aa)	Surface accessibility by antibody binding
  Induction conditions	Varied time/temperature combinations	Total expression level

• Quantitative assessment methods:

  • Enzyme-linked immunosorbent assay (ELISA) to quantify surface-accessible protein

  • Fluorescence-activated cell sorting (FACS) to measure the distribution of expression levels within the population

  • Binding assays with target ligands to confirm functional surface display

Implementing these experimental approaches in a systematic manner allows researchers to identify optimal conditions for efficient surface display of UPF0291 protein lp_2062 on L. plantarum .

What emerging technologies might enhance the application potential of recombinant L. plantarum UPF0291 protein lp_2062?

Several emerging technologies hold promise for expanding the application potential of recombinant L. plantarum UPF0291 protein lp_2062:

• CRISPR-Cas9 genome editing:

  • Direct chromosomal integration of lp_2062 expression cassettes for increased stability

  • Precise modification of native regulatory elements to enhance expression

  • Knockout of competing pathways to redirect cellular resources toward target protein production

• Synthetic biology approaches:

  • Design of synthetic promoters and ribosome binding sites optimized for lp_2062 expression

  • Development of genetic circuits for auto-inducible or environmentally responsive expression

  • Construction of minimal genome L. plantarum strains with reduced metabolic burden

• Advanced bioinformatics for expression optimization:

  • Machine learning algorithms to predict optimal codon usage patterns

  • Protein structure prediction tools to guide stability-enhancing modifications

  • Systems biology modeling of metabolic networks to identify bottlenecks in protein production

• Innovative delivery systems:

  • Encapsulation technologies to protect recombinant L. plantarum during gastrointestinal transit

  • Biofilm-based delivery systems for sustained release applications

  • Mucoadhesive formulations to enhance residence time at mucosal surfaces

• Expanded application domains:

  • Development of lp_2062 fusion proteins with novel functional domains

  • Application in metabolic engineering as scaffold proteins

  • Integration into biosensor systems for environmental or biomedical monitoring

These technologies represent promising avenues for enhancing the expression, stability, delivery, and application potential of recombinant L. plantarum UPF0291 protein lp_2062 in both research and applied contexts .

How might recombinant L. plantarum UPF0291 protein lp_2062 be adapted for therapeutic applications?

Adapting recombinant L. plantarum UPF0291 protein lp_2062 for therapeutic applications involves several strategic considerations:

• Immunomodulatory applications:

  • Fusion with immunogenic epitopes to develop oral or mucosal vaccines

  • Development of prime-boost-boost immunization protocols (e.g., administration on days 1-3, 14-16, and 29-31)

  • Assessment of mucosal, cellular, and systemic immune responses to determine efficacy

• Protein delivery optimization:

  • Enhancement of gastrointestinal survival through selection of robust L. plantarum strains with high tolerance to acid (pH 3) and bile salts (0.3%)

  • Evaluation of adhesion to intestinal epithelial cells using in vitro models like Caco-2 cells

  • Confirmation of in vivo transit time and colonization patterns through fluorescent imaging techniques

• Safety and regulatory considerations:

  • Selection of L. plantarum strains with established safety profiles or GRAS (Generally Recognized As Safe) status

  • Development of biological containment strategies

  • Implementation of antibiotic resistance marker-free expression systems

• Production process development:

  • Scale-up of fermentation processes while maintaining expression efficiency

  • Development of preservation methods that maintain viability and expression capacity

  • Establishment of quality control protocols for consistent product characteristics

• Clinical evaluation pathway:

  • Preclinical efficacy studies in appropriate animal models

  • Safety assessment through toxicology studies

  • Design of first-in-human clinical trials with appropriate endpoints and biomarkers

The development of recombinant L. plantarum strains expressing the SARS-CoV-2 spike protein and swIAV HA1 antigen provides valuable precedents for developing UPF0291 protein lp_2062 for therapeutic applications, particularly in the context of mucosal vaccination or protein delivery .