Recombinant Lactobacillus plantarum Probable GTP-binding protein EngB (engB)

What makes Lactobacillus plantarum an advantageous host for recombinant protein expression?

Lactobacillus plantarum offers several advantages as an expression host, particularly for applications requiring food-grade or probiotic delivery systems. The species exhibits remarkable ecological and metabolic flexibility, allowing it to thrive in various environments . As a lactic acid bacterium, it has GRAS (Generally Recognized As Safe) status, making it suitable for food applications and oral administration . L. plantarum also possesses intrinsic immunomodulatory properties that can function as natural adjuvants for delivered antigens . Additionally, the availability of well-characterized expression systems like pSIP allows for controlled, inducible expression of heterologous proteins .

What are the main expression systems available for recombinant protein production in L. plantarum?

The pSIP expression system is one of the most widely used for recombinant protein expression in L. plantarum. This system is based on the sakacin P operon of L. sakei and includes variants like pSIP403 and pSIP409, which differ in their bacteriocin promoters (PsppA and PsppQ, respectively) . The system provides tight control of expression through induction with peptide pheromones (SppIP) . Other expression approaches include surface display systems using anchoring motifs like pgsA and signal peptides like the endogenous signal peptide 1320 (ALX04_001320) , which allow proteins to be displayed on the bacterial surface or secreted into the medium.

How do you optimize growth conditions for recombinant L. plantarum?

Optimizing growth conditions for recombinant L. plantarum involves careful consideration of several parameters:

• Culture pH: This significantly impacts recombinant protein production, with optimal pH typically between 6.0-6.5 .

• Growth temperature: Usually maintained around 30°C for L. plantarum strains, though expression can occur at 37°C .

• Media composition: Simplified MRS medium is commonly used, containing glucose, peptone, yeast extract, K₂HPO₄, MgSO₄, and MnSO₄ .

• Glucose concentration: High glucose levels can repress some promoters, necessitating controlled feeding strategies .

• Induction parameters: For pSIP systems, inducer concentration (typically 50 ng/mL SppIP) and induction timing (usually mid-log phase) are critical .

Under optimized conditions, recombinant protein yields can reach 70% of the total soluble intracellular protein of the host organism .

What are the critical factors to consider when designing expression constructs for L. plantarum?

When designing expression constructs for L. plantarum, several critical factors should be considered:

• Codon optimization: Adapting the coding sequence to the codon usage preference of L. plantarum significantly improves expression levels .

• Promoter selection: For inducible expression, the pSIP system offers controllable promoters with different basal expression levels (PsppA vs PsppQ) .

• Signal peptides: For secreted or surface-displayed proteins, appropriate signal peptides must be selected, such as the endogenous signal peptide 1320 .

• Anchoring domains: For surface display, anchoring motifs like pgsA can efficiently display proteins on the bacterial surface .

• Affinity tags: Including tags like 6×His facilitates purification and detection of recombinant proteins .

• Terminator sequences: Proper terminator sequences ensure stable mRNA and efficient translation .

The design strategy should be tailored to the specific properties and intended application of the target protein, with consideration of protein size, folding requirements, and functional domains.

How can protein stability be assessed in L. plantarum expression systems?

Protein stability in L. plantarum expression systems can be assessed through multiple approaches:

• Environmental stability testing: Subjecting the recombinant bacteria to various conditions to evaluate protein retention:

  • Temperature stability (e.g., 37°C, 50°C for 20 minutes)

  • pH stability (e.g., pH 1.5 to 7.0 for 30 minutes)

  • Bile salt exposure (0.2% to 0.5% for 2 hours)

• Analytical methods:

  • Western blotting to detect protein integrity over time

  • Flow cytometry to quantify surface-displayed protein retention

  • Enzyme activity assays for functional stability (if applicable)

  • Circular dichroism to confirm structural integrity

• In vivo stability:

  • For vaccine applications, stability during gastrointestinal transit

  • Persistence of recombinant bacteria in animal models

  • Duration of antigen presentation and immune response induction

Stability assessment should incorporate both structural and functional analyses to ensure the recombinant protein maintains its intended activity.

What strategies can be employed to enhance protein expression levels in L. plantarum?

Several strategies can enhance protein expression levels in L. plantarum:

• Ortholog screening: Testing homologous proteins from different bacterial species can identify variants with superior expression characteristics. In one study, screening five RseP orthologs revealed that the E. faecium variant yielded the highest expression levels in L. plantarum, while the native L. plantarum variant showed lower production .

• Fermentation optimization:

  • Fed-batch cultivation can prevent glucose repression

  • Controlling pH at 6.0-6.5 improves productivity

  • Optimizing induction timing based on growth phase

  • Temperature modulation during expression phase

• Vector engineering:

  • Using stronger ribosome binding sites

  • Incorporating transcription terminators

  • Selecting appropriate promoter strength

  • Optimizing the signal peptide sequence for the specific protein

• Host strain engineering:

  • Knockout of proteases that might degrade the recombinant protein

  • Modification of metabolic pathways to enhance precursor availability

  • Adapting the strain for specific expression requirements

Under optimal conditions, recombinant protein yields can reach up to 200 mg per liter of fermentation medium, representing approximately 70% of the total soluble intracellular protein .

How can L. plantarum be engineered as a biosensor or targeted delivery system?

L. plantarum can be engineered as sophisticated biosensor and targeted delivery systems through several advanced approaches:

• Quorum sensing-based detection systems: Engineered L. plantarum can incorporate bacterial quorum sensing systems like the AgrQS system to detect pathogen-specific signals. For example, L. plantarum WCSF I has been engineered to detect autoinducing peptides (AIPs) from Staphylococcus aureus, with activation strengths increasing from 1.2-fold to 5.3-fold through promoter engineering .

• Conditional expression systems:

  • Pathogen-responsive promoters that activate upon detection of specific microbial signatures

  • Environment-sensitive expression that responds to gut conditions

  • Host cell targeting via surface receptors

• Bifunctional systems that combine detection and response:

  • Detection of S. aureus through AIP sensing coupled with expression of antimicrobial lysostaphin

  • Sensing of enteric pathogens coupled with immunomodulatory or antimicrobial responses

  • Surface display of targeting moieties combined with therapeutic protein expression

These systems can be fine-tuned for sensitivity and specificity through careful selection of sensing elements and optimization of the genetic circuits controlling response mechanisms.

What immunological considerations are important when using L. plantarum for vaccine delivery?

When using L. plantarum for vaccine delivery, several immunological considerations are crucial:

• Immune response profile:

  • L. plantarum expressing heterologous antigens can induce both systemic (IgG) and mucosal (sIgA) antibody responses

  • Recombinant L. plantarum strains can stimulate cellular immune responses, including CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells

  • The species can enhance dendritic cell maturation and cytokine production (IL-6, IL-12, TNF-α)

• Adjuvant effects and enhancement strategies:

  • Co-expression of immunostimulatory molecules like IL-33 or CTA1-DD can significantly enhance immune responses

  • Surface proteins like GAPDH can improve adhesion to epithelial cells, potentially enhancing antigen delivery

  • Pattern recognition receptor engagement, particularly TLR2/TLR6 heterodimers, contributes to immunomodulatory capacity

• Quantitative measurement of immune responses:

  • Antibody titers (S/P values > 0.6 often considered positive)

  • Flow cytometry to assess T cell subset differentiation

  • ELISA to measure cytokine production and sIgA levels

  • Assessment of dendritic cell maturation markers (CD80, CD86)

Data from studies shows that L. plantarum-based vaccines can achieve significant protection. For example, recombinant L. plantarum expressing ALV-J gp85 provided protection against viremia challenge, with vaccination groups showing significantly lower positive viremia ratios compared to control groups .

How can researchers troubleshoot low expression or misfolding of complex proteins in L. plantarum?

Troubleshooting low expression or misfolding of complex proteins in L. plantarum requires a systematic approach:

• Expression vector analysis:

  • Verify the integrity of the expression construct by sequencing

  • Confirm promoter functionality using a reporter gene

  • Test different signal peptides if secretion or surface display is intended

  • Consider the compatibility of the protein with the fusion tags or anchoring domains

• Protein-specific optimizations:

  • Screen homologous proteins from different species (ortholog screening)

  • Modify the protein by removing problematic domains or creating fusion constructs

  • Introduce mutations to improve stability without affecting function

  • Co-express chaperones or foldases to assist proper folding

• Expression condition optimization matrix:

  Parameter	Test Range	Monitoring Method
  Induction timing	OD600 0.3-1.0	Western blot/activity
  Inducer concentration	10-100 ng/mL	Western blot/activity
  Temperature	20-37°C	Western blot/activity
  pH	5.5-7.0	Western blot/activity
  Media composition	Various supplements	Western blot/activity

• Analytical techniques for protein quality assessment:

  • Circular dichroism to assess secondary structure

  • Size exclusion chromatography to evaluate oligomeric state

  • Functional assays specific to the protein of interest

  • Proteomic analysis to identify potential degradation products

One successful example involves the expression of EfmRseP in L. plantarum, which yielded approximately 1 mg of pure protein per 3 g of wet-weight cell pellet after optimization, while the same protein failed to express solubly in two of three E. coli strains tested .

What are the most effective purification strategies for recombinant proteins from L. plantarum?

Purifying recombinant proteins from L. plantarum typically involves these key steps:

• Cell disruption methods:

  • For intracellular proteins: sonication, bead beating, or enzymatic lysis

  • For surface-displayed proteins: extraction with LiCl (5M) or guanidine-HCl

  • For secreted proteins: precipitation from culture supernatant

• Primary capture:

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

  • Affinity chromatography based on the specific tag used (e.g., GST, MBP)

  • Ion exchange chromatography as an alternative first step

• Secondary purification:

  • Size exclusion chromatography to separate oligomeric states and remove aggregates

  • Ion exchange chromatography for further purification

  • Hydrophobic interaction chromatography for certain proteins

• Special considerations for membrane proteins:

  • Inclusion of appropriate detergents during extraction and purification

  • Validation of protein structural integrity using circular dichroism

  • Assessment of homogeneity by analytical size exclusion chromatography

A successful example is the purification of RseP from E. faecium expressed in L. plantarum, which yielded approximately 1 mg of pure protein per 3 g of wet-weight cell pellet using IMAC followed by size-exclusion chromatography .

How can researchers assess the functional activity of recombinant proteins expressed in L. plantarum?

Assessing the functional activity of recombinant proteins expressed in L. plantarum depends on the protein type but generally includes:

• Enzymatic activity assays:

  • For enzymes like β-galactosidase, specific activity can be measured (e.g., 190 U/mg for purified L. reuteri β-galactosidase)

  • Activity comparison to native or commercially available equivalents

  • Kinetic parameter determination (Km, Vmax, kcat)

• Binding and interaction assays:

  • Surface plasmon resonance for protein-protein interactions

  • ELISA-based binding assays

  • Cell adhesion assays for surface-displayed proteins (e.g., GAPDH-mediated adhesion)

• In vitro biological activity:

  • For antimicrobial proteins: inhibition zone assays against target pathogens

  • For immunomodulatory proteins: cytokine induction in immune cell cultures

  • For receptor proteins: ligand binding assays

• In vivo functional validation:

  • Animal models appropriate for the protein function

  • Immune response measurement for vaccine antigens

  • Pathogen challenge studies to assess protection

Specific examples include testing bacteriocin receptor functionality through sensitivity assays to specific bacteriocins , or evaluating vaccine antigen immunogenicity through antibody titer measurements in immunized animals .

What analytical methods are most useful for characterizing recombinant proteins produced in L. plantarum?

Multiple analytical methods are essential for comprehensive characterization of recombinant proteins from L. plantarum:

• Primary structure verification:

  • Mass spectrometry for molecular weight confirmation and peptide mapping

  • N-terminal sequencing to confirm proper processing

  • Western blotting with specific antibodies for identity confirmation

• Expression level quantification:

  • Densitometry analysis of SDS-PAGE gels

  • ELISA-based quantification

  • Flow cytometry for surface-displayed proteins (provides data on percentage of expressing cells)

• Structural integrity assessment:

  • Circular dichroism for secondary structure analysis

  • Size exclusion chromatography for oligomeric state determination

  • Thermal shift assays for stability assessment

• Localization confirmation:

  • Immunofluorescence microscopy for surface-displayed proteins

  • Transmission electron microscopy to visualize surface structures

  • Cell fractionation followed by Western blotting

• Stability analysis:

  • Environmental challenge tests (pH, temperature, bile salts)

  • Storage stability at different temperatures

  • Functional activity retention over time

In one study characterizing recombinant SARS-CoV-2 spike protein expressed in L. plantarum, a combination of Western blotting, transmission electron microscopy, immunofluorescence assay, and flow cytometry confirmed successful surface display with approximately 37.5% positive rate of expression .

How can L. plantarum expression systems be used for structural biology studies of membrane proteins?

L. plantarum expression systems offer unique advantages for structural biology studies of membrane proteins:

• Expression capabilities:

  • L. plantarum can express complex membrane proteins that fail in traditional systems like E. coli

  • The pSIP expression system provides tight control, allowing optimization for proper membrane protein folding

  • Ortholog screening can identify variants with superior expression and stability

• Membrane environment considerations:

  • The Gram-positive cell envelope provides a distinct lipid environment that may benefit certain membrane proteins

  • L. plantarum offers an alternative membrane composition that can be advantageous for proteins that misfold in E. coli

  • Extraction protocols can be optimized to maintain native-like lipid interactions

• Purification strategies for structural studies:

  • Detergent screening specific to L. plantarum-expressed membrane proteins

  • Nanodiscs or other membrane mimetics for stabilization

  • Scale-up capabilities for obtaining sufficient quantities for structural studies

A significant example is the successful expression and purification of the integral membrane protein RseP from E. faecium, which yielded approximately 1 mg of pure protein per 3 g of wet-weight cell pellet. This protein failed to express solubly in multiple E. coli strains, highlighting the potential of L. plantarum for challenging membrane proteins .

What are the latest developments in using engineered L. plantarum for targeted therapeutics?

Recent developments in engineered L. plantarum for targeted therapeutics showcase sophisticated approaches:

• Pathogen-sensing therapeutic systems:

  • Engineering L. plantarum WCSF I with quorum sensing genetic circuits to detect Staphylococcus aureus through AIP sensing

  • Coupling detection mechanisms with therapeutic protein expression (e.g., lysostaphin) for targeted antimicrobial activity

  • Achieving 5.3-fold activation strength through optimized promoter engineering

• Cancer immunotherapy applications:

  • L. plantarum expressing tumor antigens like NY-ESO-1 can induce specific antibodies and T-cell responses

  • Surface display of tumor antigens on L. plantarum induces dendritic cell maturation and cytokine production

  • These systems can stimulate both IL-12 and TNF-α production while not inducing IL-4, suggesting a favorable Th1-biased response

• Oral vaccine innovations:

  • Surface display systems using anchoring motifs like pgsA for efficient antigen presentation

  • Combination with adjuvant molecules like IL-33 or CTA1-DD to enhance immune responses

  • Demonstrations of both mucosal (sIgA) and systemic (IgG) immunity induction

• Biocontainment and controlled delivery:

  • Engineered systems for controlled protein release in specific environments

  • Signal peptide-guided delivery systems that respond to environmental cues

  • Biosafety enhancements to prevent environmental spread

These developments represent a paradigm shift from simple protein expression to sophisticated sensing-response systems with potential applications in infectious disease, cancer, and autoimmune disorders.

What computational approaches can predict protein expression success in L. plantarum?

Computational approaches for predicting protein expression success in L. plantarum include:

• Codon optimization algorithms:

  • Analysis of L. plantarum-specific codon usage bias

  • Optimization of codons while maintaining mRNA secondary structure stability

  • Identification and removal of detrimental rare codon clusters

• Signal peptide and secretion prediction:

  • Algorithms to evaluate signal peptide compatibility with the L. plantarum secretion machinery

  • Prediction of protein-specific secretion barriers

  • Modeling of signal peptide-protein interactions that might affect folding

• Protein structural feature analysis:

  • Identification of features associated with poor expression (hydrophobic patches, aggregation-prone regions)

  • Prediction of disulfide bond formation potential in the L. plantarum intracellular environment

  • Modeling of protein stability in the context of L. plantarum cytoplasmic conditions

• Expression system optimization models:

  • Statistical design of experiments (DoE) approaches for expression parameter optimization

  • Response surface methodology to identify optimal expression conditions

  • Machine learning models trained on expression data from multiple proteins

An example application is the optimization of L-ribulose production using central composite designs, which predicted an optimal operation point with an L-arabinose concentration of 100 g/L, a borate concentration of 500 mM, and a temperature of 48°C. This computational approach predicted an initial production rate of 29.1 g/L/h and a conversion of L-arabinose to L-ribulose of 0.70 mol/mol .