Recombinant Lactobacillus plantarum Enolase 2 (eno2)

What is L. plantarum Enolase 2 and how does it differ from Enolase 1?

L. plantarum contains two genes encoding alpha-enolases: enoA1 (lp_0792) and enoA2 (lp_1920). Both genes are expressed under standard growth conditions and encode proteins with predicted alpha-enolase activity. EnoA1 (48 kDa, pI 4.6) belongs to the central glycolytic genes operon (cggR), while EnoA2 (46.6 kDa, pI 4.9) is encoded elsewhere in the genome. The two proteins share significant sequence identity but appear to have distinct functions beyond their enzymatic role in glycolysis . While EnoA1 has been identified as a fibronectin-binding protein associated with the cell surface, the specific non-glycolytic functions of EnoA2 remain less characterized.

What genomic organization characterizes the enoA2 gene in L. plantarum?

Unlike enoA1, which is part of the pentacistronic cggR operon containing genes encoding central glycolytic enzymes, enoA2 is not located within this operon . The genomic context of enoA2 differs from that of enoA1, suggesting possible distinct regulation and function. This genomic organization is similar to what has been observed in other Lactobacillus species, such as L. johnsonii, where multiple enolase genes (eno1-3) are present but located in different chromosomal regions .

What expression systems are optimal for recombinant L. plantarum Enolase 2 production?

E. coli expression systems have been successfully used for recombinant production of bacterial enolases, including those from Lactobacillus species. For L. plantarum Enolase 2, a common approach involves cloning the enoA2 gene into expression vectors containing a His6-tag fusion to facilitate purification. Expression can be optimized using E. coli BL21(DE3) or similar strains with IPTG induction under controlled temperature (typically 25-30°C) to enhance soluble protein yield . The recombinant protein can be expressed with either N- or C-terminal His-tags, though care should be taken if studying plasminogen binding activity, as C-terminal lysine residues can influence this interaction.

What purification strategy yields the most active recombinant Enolase 2?

Purification of His6-tagged L. plantarum Enolase 2 is typically performed by affinity chromatography under non-denaturing conditions to preserve enzyme activity. After cell lysis (typically using sonication or French press), the clarified lysate is applied to a nickel-charged affinity resin (Ni-NTA or similar) and washed with buffers containing low concentrations of imidazole to reduce non-specific binding. The protein is then eluted with higher imidazole concentrations (150-300 mM) . For highest purity, a second purification step such as ion-exchange chromatography or size exclusion chromatography may be employed. The purified enzyme should be dialyzed against a suitable buffer (often containing Mg2+, which is important for enolase activity) and stored at -80°C in small aliquots to maintain activity.

How can the oligomeric state of recombinant Enolase 2 be verified?

Bacterial enolases, including those from Lactobacillus species, typically exist as octamers with a molecular weight of approximately 370-380 kDa. The oligomeric state of purified recombinant L. plantarum Enolase 2 can be verified using size exclusion chromatography, analytical ultracentrifugation, or native PAGE. Evidence from related enolases suggests that the oligomeric structure is important for both enzymatic function and non-enzymatic roles such as plasminogen binding . For instance, L. crispatus enolase has been shown to exist as an octamer both in its native form in the extracellular proteome and as a recombinant His6-protein purified from E. coli .

What methods can be used to assess the glycolytic activity of recombinant Enolase 2?

The enzymatic activity of recombinant L. plantarum Enolase 2 can be assessed using a coupled reaction with 2-phosphoglycerate (2-PGE) as a substrate. The standard assay measures the conversion of 2-PGE to phosphoenolpyruvate (PEP) by monitoring the increase in absorbance at 240 nm, which corresponds to the formation of PEP . Alternatively, a coupled enzyme assay can be used, linking enolase activity to pyruvate kinase and lactate dehydrogenase reactions, measuring NADH oxidation at 340 nm. The specific activity is typically expressed as μmol of substrate converted per minute per mg of protein under standard conditions (pH 6.5-7.5, 25-37°C). For accurate results, it's crucial to include Mg2+ in the assay buffer, as it's required for enolase enzymatic activity.

How does recombinant L. plantarum Enolase 2 compare to Enolase 1 in terms of enzymatic properties?

Both EnoA1 and EnoA2 from L. plantarum demonstrate enolase enzymatic activity, converting 2-PGE to PEP. While specific kinetic parameters comparing the two enzymes in L. plantarum have not been extensively documented, studies with other Lactobacillus species have shown that multiple enolase variants can exhibit different catalytic efficiencies. The differences in enzymatic properties may relate to their distinct physiological roles. Based on studies of His6-enolases from other bacteria, it's expected that both enzymes would be catalytically active but might show differences in specific activity, substrate affinity (Km), or response to environmental factors such as pH and temperature .

What are the key structural features of L. plantarum Enolase 2 that contribute to its function?

L. plantarum Enolase 2, like other bacterial enolases, likely possesses conserved structural domains essential for its glycolytic function, including catalytic sites and Mg2+-binding sequences. The protein is expected to form an octameric structure with a molecular weight of approximately 370-380 kDa, composed of identical subunits of about 46.6 kDa . While specific structural data for L. plantarum Enolase 2 is limited, comparative sequence analysis with better-characterized enolases can identify conserved catalytic residues and potential functional motifs. Unlike some bacterial enolases that possess C-terminal lysines important for plasminogen binding, L. plantarum Enolase 2 and other lactobacillar enolases may utilize internal lysine, arginine, or histidine residues for this function .

How can the surface localization of Enolase 2 be investigated?

Surface localization of L. plantarum Enolase 2 can be investigated using multiple complementary approaches. Immune electron microscopy using anti-enolase antibodies followed by secondary antibodies conjugated with colloidal gold particles can visualize the surface distribution of the protein, as demonstrated for EnoA1 in L. plantarum LM3 . Surface protein extraction followed by two-dimensional electrophoresis and Western blotting with anti-enolase antibodies can confirm the presence of Enolase 2 in the cell wall fraction. Additionally, flow cytometry with fluorescently-labeled antibodies can provide quantitative data on surface expression levels under different growth conditions. It's worth noting that the surface localization of enolases in Lactobacillus appears to be pH-dependent, with more efficient release into the medium at neutral pH compared to attachment to the cell wall at acidic pH .

What methods can be used to identify potential binding partners of recombinant Enolase 2?

Several techniques can identify potential binding partners of recombinant L. plantarum Enolase 2. For extracellular matrix interactions, solid-phase binding assays with immobilized fibronectin, laminin, collagen, or other matrix proteins can be used, followed by detection with anti-His tag or anti-enolase antibodies . Pull-down assays using His-tagged Enolase 2 as bait, followed by mass spectrometry, can identify protein-protein interactions. Surface plasmon resonance (SPR) can provide quantitative binding parameters. For plasminogen binding, an overlay immunoblotting assay can be employed, where proteins separated by SDS-PAGE are transferred to membranes, incubated with plasminogen, and detected with anti-plasminogen antibodies . Additionally, far-Western blotting techniques can be used to screen for novel protein interactions.

How does L. plantarum Enolase 2 compare to enolases from other Lactobacillus species?

L. plantarum Enolase 2 shares sequence and functional similarities with enolases from other Lactobacillus species, though with distinct characteristics. In L. johnsonii, three enolase genes (eno1-3) have been identified, with differential expression patterns and varying degrees of plasminogen binding efficiency . L. johnsonii enolases 1 and 2 efficiently bind plasminogen and enhance its activation, similar to what is observed with L. crispatus enolase, while L. johnsonii enolase 3 shows lower activity . Comparative sequence analysis reveals that lactobacillar enolases typically lack C-terminal lysines that are present in some pathogenic streptococcal enolases and contribute to plasminogen binding, suggesting they employ different binding mechanisms . The table below summarizes key features of enolases from different Lactobacillus species:

Can sequence analysis predict functional differences between L. plantarum Enolase 2 and enolases from other species?

Sequence analysis can indeed provide insights into potential functional differences between L. plantarum Enolase 2 and other bacterial enolases. Key features to examine include:

• Internal plasminogen-binding sites: While some streptococcal enolases contain the internal binding motif 248FYDKERKVY where lysines and glutamic acid are important for plasminogen binding, L. crispatus enolase contains a related but distinct sequence (248FYNKDDHKY) . The sequence in Staphylococcus aureus enolase (250FYENGVYDY) lacks basic residues in this region yet maintains high plasminogen-binding activity, suggesting that other residues are involved .

• C-terminal lysines: Many plasminogen-binding proteins, including some bacterial enolases, contain C-terminal lysines that interact with plasminogen kringle domains. Lactobacillar enolases typically lack these C-terminal lysines but still bind plasminogen efficiently .

• Surface-exposed basic residues: Recent studies suggest that arginine and histidine residues can also contribute to plasminogen binding . Mapping these residues on predicted protein structures can identify potential binding interfaces.

• ECM-binding domains: Comparison with known adhesins can reveal potential motifs involved in binding to fibronectin, laminin, or collagen.

Detailed sequence alignment and structural modeling can predict functional sites and guide targeted mutagenesis experiments to verify their role.

How can recombinant Enolase 2 be used to study L. plantarum adhesion to host tissues?

Recombinant L. plantarum Enolase 2 can serve as a valuable tool for studying bacterial adhesion to host tissues through several experimental approaches:

These approaches provide complementary data on the role of Enolase 2 in L. plantarum adhesion to host tissues.

What role might Enolase 2 play in the probiotic properties of L. plantarum?

Enolase 2, as a potential surface protein in L. plantarum, may contribute to the probiotic properties of this bacterium through several mechanisms:

• Adhesion to intestinal epithelium: If Enolase 2 functions as an adhesin like EnoA1, it may facilitate colonization of the gastrointestinal tract, an important property for probiotics.

• Interaction with extracellular matrix: Binding to components like fibronectin, laminin, or collagen may help L. plantarum interact with the gut mucosal layer.

• Immune modulation: Surface proteins of probiotic bacteria can interact with immune cells and potentially modulate immune responses.

• Competitive exclusion: By occupying binding sites, L. plantarum surface proteins may reduce adhesion of pathogenic bacteria.

• Plasminogen system interaction: The ability of lactobacillar enolases to interact with the host plasminogen system may serve functions distinct from those in pathogenic bacteria, potentially contributing to mucosal homeostasis .

How can the pH-dependent localization of Enolase 2 be studied in the context of gastrointestinal transit?

Studies have shown that the surface localization of enolases in lactobacilli can be pH-dependent, with more efficient release into the medium at neutral pH compared to attachment to the cell wall at acidic pH . This property is particularly relevant in the context of gastrointestinal transit, where bacteria encounter varying pH environments. To study this phenomenon with L. plantarum Enolase 2:

• In vitro pH shift experiments: Expose L. plantarum cultures to sequential pH changes mimicking gastrointestinal transit (acidic stomach to neutral/slightly alkaline intestine) and analyze the distribution of Enolase 2 between cell surface and culture supernatant.

• Surface protein extraction at different pH: Comparative analysis of surface proteins extracted at various pH values using techniques like two-dimensional electrophoresis and Western blotting with anti-enolase antibodies.

• Immunofluorescence microscopy: Visualize the surface distribution of Enolase 2 under different pH conditions using fluorescently labeled antibodies.

• In vivo transit studies: Recover L. plantarum from different segments of the gastrointestinal tract in animal models and analyze the surface expression of Enolase 2.

• Simulated gastrointestinal conditions: Use in vitro models that simulate the dynamic conditions of the gastrointestinal tract, including pH changes, bile salts, and digestive enzymes.

These approaches can provide insights into how the localization of Enolase 2 changes during gastrointestinal transit and the potential implications for host-microbe interactions.

What strategies can address poor expression or solubility of recombinant Enolase 2?

Several strategies can improve expression and solubility of recombinant L. plantarum Enolase 2:

• Expression conditions optimization: Test different temperatures (16-30°C), IPTG concentrations (0.1-1.0 mM), and induction times (4-24 hours) to identify conditions that favor soluble protein production.

• Expression vectors: Try different fusion tags (His, GST, MBP, SUMO) which can enhance solubility. MBP and SUMO tags are particularly effective for improving solubility.

• Host strain selection: Test multiple E. coli strains (BL21(DE3), Rosetta, Arctic Express) designed for enhanced protein expression or folding.

• Co-expression with chaperones: Systems that co-express molecular chaperones (GroEL/ES, DnaK/J) can improve folding and solubility.

• Buffer optimization: During purification, screen different buffers, salt concentrations, and additives (glycerol, detergents, arginine) that can enhance protein stability and solubility.

• Refolding strategies: If expression yields insoluble inclusion bodies, develop a refolding protocol using gradual dialysis or on-column refolding.

• Codon optimization: Synthesize a codon-optimized gene for expression in E. coli to address potential codon bias issues.

Systematic testing of these approaches can significantly improve recombinant protein yield and quality.

How can enzymatic activity be preserved during purification and storage?

Preserving the enzymatic activity of recombinant L. plantarum Enolase 2 requires careful attention during purification and storage:

• Buffer composition: Include essential cofactors like Mg2+ in purification and storage buffers, as they are critical for enolase activity.

• Reducing agents: Add DTT or β-mercaptoethanol (1-5 mM) to prevent oxidation of cysteine residues that might be important for catalysis or structure.

• Stabilizing agents: Include glycerol (10-20%) or sucrose in storage buffers to stabilize protein structure.

• pH optimization: Determine the optimal pH for stability (typically pH 7.0-8.0) and maintain buffers in this range.

• Temperature control: Perform all purification steps at 4°C and avoid freeze-thaw cycles by storing the protein in small aliquots.

• Protease inhibitors: Include a protease inhibitor cocktail during initial extraction steps to prevent degradation.

• Activity assays: Monitor enzymatic activity throughout the purification process to identify steps where activity loss occurs.

• Storage conditions: For long-term storage, flash-freeze small aliquots in liquid nitrogen and store at -80°C rather than -20°C.

Following these guidelines can significantly improve the stability and retention of enzymatic activity in purified recombinant Enolase 2.

What controls are essential for plasminogen binding and activation experiments?

Proper controls are crucial for reliable plasminogen binding and activation experiments with recombinant L. plantarum Enolase 2:

• Negative controls:

  • Buffer-only controls without the recombinant protein

  • Unrelated proteins of similar size and charge (e.g., BSA)

  • Heat-denatured Enolase 2 to distinguish between specific and non-specific binding

• Positive controls:

  • Well-characterized plasminogen-binding proteins (e.g., staphylococcal or streptococcal enolases)

  • Commercial plasminogen activators (tPA, uPA) for activation assays

• Specificity controls:

  • Lysine analog ε-aminocaproic acid (EACA) to inhibit binding via lysine residues

  • Dose-dependent competition with soluble plasminogen

  • Anti-enolase antibodies to block specific binding sites

• Enzymatic activity controls:

  • Plasmin-specific chromogenic or fluorogenic substrates

  • Plasmin inhibitors (e.g., α2-antiplasmin)

  • Controls without plasminogen activators (tPA or uPA)

• Technical controls:

  • Multiple technical and biological replicates

  • Different batches of recombinant protein

  • Variation in experimental conditions (pH, ionic strength)

Including these controls ensures that observed plasminogen binding and activation are specific properties of Enolase 2 rather than experimental artifacts.