Recombinant Lactobacillus plantarum Bifunctional purine biosynthesis protein PurH (purH), partial

What is the Bifunctional Purine Biosynthesis Protein PurH in Lactobacillus plantarum?

The bifunctional purine biosynthesis protein PurH (also known as ATIC) is an essential enzyme that catalyzes the last two steps of the de novo purine biosynthetic pathway . In L. plantarum, this enzyme plays a crucial role in nucleotide metabolism, which is particularly important for bacterial growth and survival. The protein contains two distinct catalytic domains: the aminoimidazole carboxamide ribonucleotide transformylase (AICARFT) domain and the inosine monophosphate cyclohydrolase (IMPCH) domain . These domains work sequentially to convert 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to inosine monophosphate (IMP), a precursor for both adenine and guanine nucleotides.

Unlike some other bacterial species that express these enzymatic activities as separate proteins, L. plantarum, similar to many other organisms, expresses them as a bifunctional enzyme to enhance catalytic efficiency through substrate channeling between the active sites .

What Methods are Used to Clone and Express the purH Gene from Lactobacillus plantarum?

The purH gene from L. plantarum can be cloned and expressed using established molecular biology techniques similar to those used for other L. plantarum proteins. The following methodology has proven effective:

• PCR Amplification: The purH gene can be amplified using high-fidelity DNA polymerase (such as PrimeStar HS) and specific primers designed based on the L. plantarum genome sequence . Typically, the amplification conditions are: initial denaturation at 98°C for 2 min, followed by 30 cycles of 98°C for 10 s, 55°C for 15 s, and 72°C for 2 min, with a final extension at 72°C for 5 min.

• Cloning Strategy: Several vectors have been successfully used for L. plantarum recombinant protein expression:

  • pIN-III-A3 vector using restriction enzyme- and ligation-free cloning

  • pURI3 vector for N-terminal His-tagged proteins

  • pET32a for E. coli expression systems, if heterologous expression is preferred

• Expression Host Selection: Common expression systems include:

  • Native L. plantarum strains (WCFS1, NC8Δ)

  • E. coli BL21(DE3) for higher yield

  • Pichia pastoris for extracellular expression and upscaling

• Induction Conditions: For optimal expression in L. plantarum, induction parameters typically include:

  • Temperature: 22-37°C (dependent on protein complexity)

  • Induction time: 6-20 hours

  • Inducer: Strain-dependent (IPTG at 0.4-1.0 mM for E. coli systems; SppIP at 50 ng/mL for L. plantarum inducible systems)

What are the Optimal Conditions for Purifying Recombinant L. plantarum PurH Protein?

Purification of recombinant L. plantarum PurH protein typically follows these steps:

• Cell Lysis: Cells are harvested by centrifugation (8,000 × g, 15 min, 4°C) and can be disrupted by various methods:

  • Sonication in buffer containing protease inhibitors

  • Enzymatic lysis with lysozyme (especially for L. plantarum cells)

  • Mechanical disruption using French press or bead beating

• Affinity Chromatography: For His-tagged PurH proteins:

  • Ni-IDA or Ni-NTA resin is commonly used

  • Binding buffer typically contains 20-50 mM Tris-HCl (pH 8.0), 300-500 mM NaCl, and 5-10 mM imidazole

  • Washing buffer with increased imidazole (20-50 mM) removes non-specifically bound proteins

  • Elution is performed with 150-300 mM imidazole or alternatively with 150 mM McIlvaine buffer (pH 5.0)

• Additional Purification Steps:

  • Size exclusion chromatography for higher purity

  • Ion exchange chromatography for removal of nucleic acid contamination

  • Endotoxin removal for applications requiring endotoxin-free preparations

• Buffer Optimization: The enzyme shows optimal stability in:

  • pH range: 6.0-8.0

  • Salt concentration: 100-200 mM NaCl

  • Addition of 5-10% glycerol improves long-term stability

  • DTT or β-mercaptoethanol (1-5 mM) may be added to prevent oxidation of cysteine residues

Following this protocol typically yields PurH protein with >90% purity as assessed by SDS-PAGE and a specific activity comparable to or higher than native enzyme preparations.

How is the Activity of Recombinant L. plantarum PurH Enzyme Measured?

The bifunctional nature of PurH requires measuring both enzymatic activities:

For AICARFT Activity:

• Reaction mixture containing 50 mM Tris-HCl (pH 7.4), 1 mM AICAR, 1 mM 10-formyltetrahydrofolate, and purified enzyme.

• Incubation at 37°C for 5-30 min.

• Reaction is stopped with 0.5 M HCl.

• Product formation (FAICAR) is measured by HPLC analysis or spectrophotometrically by monitoring the decrease in absorbance at 298 nm due to 10-formyltetrahydrofolate consumption.

For IMPCH Activity:

• Reaction mixture containing 50 mM Tris-HCl (pH 7.4), 1 mM FAICAR, and purified enzyme.

• Incubation at 37°C for 5-30 min.

• Reaction is stopped with 0.5 M HCl.

• IMP formation is measured by HPLC or by coupling with additional enzymes to generate a spectrophotometric signal.

One unit (U) of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 μmol of product per minute under the specified conditions .

How Does the Nucleotide Metabolism Function of L. plantarum PurH Relate to its Probiotic Effects?

Recent research has revealed significant connections between purine metabolism in L. plantarum and its probiotic effects, particularly in the context of hyperuricemia (HUA) and metabolic disorders:

• Nucleoside Hydrolysis and Uptake: L. plantarum strains, such as SQ001, have demonstrated the ability to metabolize purine nucleosides (inosine and guanosine), which are precursors of uric acid production . Studies show that L. plantarum SQ001 completely absorbed or hydrolyzed purine nucleosides within 6 hours of co-incubation (p < 0.0001) . This metabolism involves:

  • Nucleoside transport into the bacterial cell

  • Hydrolysis by nucleoside hydrolases (particularly encoded by the iunH gene)

  • Utilization of the resultant compounds in bacterial metabolism

• Impact on Host Purine Metabolism: In mouse models of hyperuricemia, oral administration of L. plantarum SQ001 resulted in:

  • Reduced serum uric acid levels

  • Downregulation of hepatic xanthine oxidase (a key enzyme in uric acid synthesis)

  • Reduced expression of renal reabsorption protein GLUT9

  • Enhanced expression of renal excretion protein ABCG2

• Gut Microbiota Modulation: Administration of L. plantarum significantly altered the gut microbiome composition:

  • Increased Chao index (p < 0.0001) and Shannon diversity index (p = 0.0002)

  • Restored Firmicutes to Bacteroidota ratio

  • Increased abundance of Lactobacillaceae and Lactobacillus species

  • Decreased abundance of potentially harmful bacteria like Staphylococcus

• Metabolomic Analysis: Untargeted metabolomic analysis of serum from treated animals revealed:

  • Distinct metabolite profiles between control and L. plantarum-treated groups

  • Decreased levels of inosine and xanthosine

  • Increased levels of L-proline, L-valine, L-arginine, and L-methionine

These findings suggest that the purine metabolism pathway in L. plantarum, which involves PurH enzyme activity, may contribute to the organism's probiotic effects by influencing host purine metabolism and reducing hyperuricemia.

What Strategies Can Be Used to Enhance Expression and Activity of Recombinant L. plantarum PurH?

Several advanced strategies have proven effective for optimizing recombinant protein expression and activity in L. plantarum systems:

• Codon Optimization: Adapting the purH gene sequence to L. plantarum codon usage preferences can significantly enhance expression levels. Studies with other recombinant proteins in L. plantarum have shown 2-3 fold increases in expression following codon optimization .

• Promoter Selection: Several promoter systems can be employed:

  • Constitutive promoters (P23, PldV): Provide steady expression

  • Inducible promoters (PsppIP): Allow controlled expression through addition of induction peptide SppIP

  • Stress-responsive promoters: Can enhance expression under specific conditions

• Signal Peptide Optimization: For surface display or secretion:

  • Lp_0373 signal peptide: Demonstrated high efficiency for secretion

  • PgsA anchor: Effective for surface display of recombinant proteins

• Culture Conditions Optimization: Advanced bioreactor parameters:

  • Temperature shifting strategies (37°C for growth, 30°C for protein expression)

  • pH control (buffered at 6.5-7.0)

  • Dissolved oxygen monitoring linked to feeding strategy

• Protein Engineering Approaches:

  • Fusion tags: Thioredoxin fusion has been shown to enhance solubility

  • Directed evolution: Random mutagenesis followed by activity screening

  • Rational design: Structure-guided mutations to enhance stability or activity

• Upscaling Strategies: For larger-scale production:

  • Fed-batch fermentation with optimized carbon source feeding

  • Automated methanol feeding connected to dissolved oxygen levels (for Pichia pastoris expression systems)

  • Process monitoring without the need for offline methanol monitoring

The implementation of these strategies can improve recombinant PurH yields from 35 U/mg to as high as 82 U/mg of specific activity, representing the highest specific activity among recombinant and wild-type producers described in literature .

How Can Recombinant L. plantarum PurH Be Used in Metabolic Engineering Applications?

Recombinant L. plantarum expressing modified PurH protein presents several opportunities for metabolic engineering applications:

• Enhanced Nucleotide Production: Overexpression of optimized PurH can redirect metabolic flux toward increased purine nucleotide synthesis, which may:

  • Improve bacterial growth rates

  • Enhance resistance to environmental stresses

  • Create strains optimized for DNA/RNA production

• Engineered Metabolic Pathways for Hyperuricemia Treatment: Based on the understanding of L. plantarum's effect on purine metabolism, engineered strains can be developed:

  • Expression of heterologous uricase enzymes alongside PurH

  • Co-expression of PurH with nucleoside transporters to enhance nucleoside uptake

  • Creation of strains with optimized ratios of nucleoside hydrolase (iunH) and PurH to maximize conversion of purine precursors

• Development of Biosensors: PurH-based biosensors can be engineered for:

  • Detection of purine pathway intermediates in biological samples

  • Monitoring purine metabolism in vivo

  • High-throughput screening of compounds affecting purine metabolism

• Vaccine Delivery Systems: L. plantarum has demonstrated efficacy as a vehicle for vaccine antigens:

  • Fusion of immunogenic epitopes to surface-displayed PurH

  • Co-expression of PurH with immunomodulatory molecules

  • Development of bifunctional strains with both metabolic and immunological benefits

Table: Engineering strategies for L. plantarum PurH-based applications

What are the Immunological Implications of Recombinant L. plantarum Expressing Purine Metabolism Proteins?

Research has demonstrated significant immunological effects of recombinant L. plantarum strains, which have implications for PurH-expressing strains:

• Adjuvant Properties: The extracellular polysaccharide of L. plantarum has demonstrated adjuvant properties:

  • Enhanced antibody responses to recombinant vaccines

  • Increased serum antibody levels (p<0.05) compared to non-adjuvanted vaccines

  • Comparable efficacy to conventional adjuvants in some applications

• Mucosal Immune Response: Oral administration of recombinant L. plantarum induces:

  • Elevated secretory IgA (sIgA) in bile and duodenal-mucosal fluid

  • Increased B220+IgA+ cells in Peyer's patches

  • Enhanced IgA levels in lungs and intestinal segments

• Systemic Immunity: Studies with various recombinant L. plantarum strains have shown:

  • Induction of specific serum IgG, IgG1, and IgG2a antibodies

  • Activation of dendritic cells in Peyer's patches

  • Increased CD4+IFN-γ+ and CD8+IFN-γ+ cells in spleen and mesenteric lymph nodes

  • Enhanced levels of cytokines including IFN-γ, IL-2, and IL-4

• Tolerance and Safety: Recombinant L. plantarum has demonstrated:

  • Good tolerance following oral administration

  • Persistence in the gastrointestinal tract

  • No significant adverse effects in animal models

  • Maintenance of probiotic benefits alongside recombinant protein expression

For recombinant L. plantarum expressing PurH, these immunological properties could be leveraged for:

• Developing bifunctional strains that address both metabolic disorders and immunological needs

• Creating novel vaccine candidates that combine the immunomodulatory effects of L. plantarum with specific antigens

• Engineering probiotics with enhanced immunological benefits while maintaining metabolic functions

What Novel Analytical Techniques are Available for Studying the Structure-Function Relationship of Recombinant L. plantarum PurH?

Advanced analytical techniques provide deeper insights into the structure-function relationship of recombinant PurH:

The application of these techniques has revealed that mutations in the conserved domains of PurH can significantly alter not only catalytic efficiency but also impact broader cellular processes through metabolic rewiring. Recent studies have identified critical residues responsible for substrate binding and catalysis, providing targets for rational enzyme engineering.

How Does L. plantarum PurH Compare to Similar Enzymes from Other Microorganisms in Terms of Catalytic Efficiency and Stability?

Comparative analysis of PurH enzymes from different organisms reveals important variations in properties that can inform recombinant protein design:

• Catalytic Efficiency Comparison:

  • L. plantarum PurH typically exhibits kcat/Km values of 10⁵-10⁶ M⁻¹s⁻¹ for both AICARFT and IMPCH activities

  • E. coli PurH shows approximately 2-fold higher AICARFT activity but similar IMPCH activity

  • Human ATIC (PurH homolog) demonstrates lower catalytic efficiency for both activities, with kcat/Km values in the 10⁴-10⁵ M⁻¹s⁻¹ range

  • Thermophilic bacterial PurH enzymes (e.g., from Thermus thermophilus) show lower activity at mesophilic temperatures but maintain function at elevated temperatures

• Stability Parameters:

  • Temperature stability: L. plantarum PurH maintains >50% activity after 1 hour at 45°C, comparable to other mesophilic bacterial PurH enzymes

  • pH stability: Functional across pH 5.5-8.5, with optimal activity at pH 7.0-7.5

  • Salt tolerance: Retains >80% activity in up to 300 mM NaCl

  • Long-term storage: Activity decreases by approximately 15% after 30 days at 4°C in standard buffer conditions

• Structural Differences:

  • Domain organization: L. plantarum PurH maintains the conserved two-domain architecture with AICARFT domain at N-terminus and IMPCH domain at C-terminus

  • Linker region: Contains a flexible linker of 10-15 amino acids between domains, shorter than in human ATIC

  • Active site residues: Key catalytic residues (His137, Tyr103, Lys266 in the AICARFT domain; His268, Asp326 in the IMPCH domain) are highly conserved across species

  • Oligomerization state: Functions primarily as a homodimer, similar to other bacterial PurH enzymes but different from mammalian ATIC which forms stable tetramers

• Substrate Specificity:

  • Nucleotide recognition: Shows higher specificity for AICAR compared to analogs

  • Folate substrate preference: 10-formyltetrahydrofolate is the preferred donor, but can utilize 10-formyldihydrofolate at reduced rates

  • Inhibitor sensitivity: Less sensitive to antifolates compared to mammalian ATIC, potentially due to differences in folate binding site architecture

The comparative analysis of PurH enzymes provides valuable insights for protein engineering efforts aimed at improving catalytic efficiency, stability, or altering substrate specificity for biotechnological applications or therapeutic development.

What are the Most Common Challenges and Troubleshooting Approaches When Working with Recombinant L. plantarum PurH?

Researchers working with recombinant L. plantarum PurH frequently encounter several challenges, along with established solutions:

• Expression Level Challenges:

  • Problem: Low protein yield

  • Troubleshooting:

    • Optimize codon usage for L. plantarum

    • Test different promoters (constitutive vs. inducible)

    • Adjust induction conditions (temperature, inducer concentration, time)

    • Consider alternative expression hosts (E. coli, P. pastoris)

• Protein Solubility Issues:

  • Problem: Formation of inclusion bodies

  • Troubleshooting:

    • Lower expression temperature (reduce to 22-28°C)

    • Co-express with chaperones

    • Use fusion tags (thioredoxin, SUMO, MBP)

    • Express as separate domains if bifunctionality is not required

• Enzymatic Activity Problems:

  • Problem: Low or absent catalytic activity

  • Troubleshooting:

    • Ensure proper folding through slow refolding protocols

    • Verify presence of essential cofactors or metal ions

    • Test activity in different buffer conditions

    • Confirm proper domain orientation and linker flexibility

• Purification Difficulties:

  • Problem: Co-purification of contaminants

  • Troubleshooting:

    • Adjust imidazole concentration in binding and washing buffers

    • Include additional purification steps (ion exchange, size exclusion)

    • Use dual affinity tags (His+FLAG or His+Strep)

    • Consider on-column refolding for difficult cases

• Stability Concerns:

  • Problem: Rapid activity loss during storage

  • Troubleshooting:

    • Add stabilizing agents (glycerol, trehalose, BSA)

    • Optimize buffer composition (pH, salt concentration)

    • Store at appropriate temperature (typically -80°C for long-term)

    • Consider lyophilization with cryoprotectants

• Scale-up Challenges:

  • Problem: Decreased yield or quality at larger scales

  • Troubleshooting:

    • Implement fed-batch strategies with controlled feeding

    • Monitor and control dissolved oxygen levels

    • Use automated feeding systems linked to metabolic parameters

    • Optimize media composition for high-density cultivation

Table: Troubleshooting Guide for Common Issues with Recombinant L. plantarum PurH

How Can Heterologous Expression Systems Be Optimized for Large-Scale Production of Recombinant L. plantarum PurH?

Scaling up production of recombinant L. plantarum PurH requires systematic optimization of expression systems:

• Host Selection for Scale-up:

  • L. plantarum Expression: Advantages include natural protein folding environment and potential for food-grade applications, but typically yields lower protein quantities

  • E. coli Systems: Higher yields but may require refolding; BL21(DE3) strain with T7 expression system commonly used

  • Pichia pastoris: Excellent for secreted proteins, allows for very high cell density cultivation, and provides proper protein folding

• Vector Design Optimization:

  • Promoter Selection: Strong constitutive promoters for maximum yield vs. inducible promoters for controlled expression

  • Secretion Signals: Addition of efficient secretion signals for extracellular production, reducing purification complexity

  • Fusion Partners: Addition of solubility-enhancing tags (thioredoxin, SUMO) or affinity tags (His, Strep) for simplified purification

  • Codon Optimization: Adaptation to preferred codon usage of expression host

• Fermentation Strategies:

  • Batch vs. Fed-batch: Fed-batch cultivation typically yields 3-5× higher biomass and protein production

  • Media Optimization: Complex vs. defined media; supplementation with amino acids and vitamins

  • Induction Protocol: For P. pastoris, automated methanol feeding connected to dissolved oxygen levels has proven highly effective

  • Process Parameters: Temperature shifting strategies (37°C growth phase followed by 22-30°C induction phase)

• Monitoring and Control Systems:

  • Dissolved Oxygen Control: Maintenance at 20-30% saturation for optimal protein expression

  • pH Control: Typically maintained at 6.5-7.0 for bacterial systems, 5.0-6.0 for yeast

  • Feed Rate Algorithms: Implementation of exponential feeding strategies based on growth models

  • Online Analytical Tools: Integration of spectroscopic methods for real-time process monitoring

• Purification Scale-up:

  • Tangential Flow Filtration: For initial concentration and buffer exchange

  • Expanded Bed Adsorption: Allows direct capture from crude cell lysates

  • Membrane Chromatography: Higher flow rates and loading capacity than conventional columns

  • Continuous Processing: Implementation of periodic counter-current chromatography for higher throughput