Recombinant Helicobacter pylori Ferrochelatase (hemH)

What is Helicobacter pylori Ferrochelatase (hemH) and what is its function?

Helicobacter pylori Ferrochelatase (hemH) is a crucial enzyme involved in the terminal step of heme biosynthesis. It catalyzes the insertion of ferrous iron into protoporphyrin IX to form heme (EC 4.99.1.1). This enzyme is also referred to as heme synthase or protoheme ferro-lyase. In H. pylori, ferrochelatase plays an essential role in cellular metabolism by enabling the bacterium to produce heme, which is required for various cellular processes including energy production and protection against oxidative stress . Understanding this enzyme's structure and function is fundamental to exploring H. pylori pathogenesis and developing targeted interventions.

How is Recombinant Helicobacter pylori Ferrochelatase produced for research use?

Recombinant H. pylori Ferrochelatase is typically produced using baculovirus expression systems. The process involves:

• Gene cloning: The hemH gene sequence is optimized for expression and cloned into an appropriate expression vector.

• Host system: Baculovirus-infected insect cells are commonly used as the expression system, providing proper folding and post-translational modifications .

• Protein expression: The recombinant protein is expressed with or without tags (the tag type is often determined during the manufacturing process).

• Purification: Multiple chromatography steps are employed to achieve high purity (>85% as assessed by SDS-PAGE) .

• Quality control: The final product undergoes various quality control measures to ensure proper folding, activity, and absence of contaminants.

This method allows for the production of functional ferrochelatase that closely resembles the native enzyme in terms of structure and activity.

What are the optimal storage and handling conditions for Recombinant Helicobacter pylori Ferrochelatase?

For optimal stability and activity of Recombinant H. pylori Ferrochelatase, researchers should follow these evidence-based storage and handling protocols:

• Long-term storage: Store at -20°C or -80°C for extended preservation. The lyophilized form has a shelf life of approximately 12 months, while the liquid form typically maintains stability for about 6 months under these conditions .

• Reconstitution procedure:

  • Centrifuge the vial briefly before opening to collect all content at the bottom

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 5-50% (50% is recommended) for cryoprotection

  • Aliquot into smaller volumes to minimize freeze-thaw cycles

• Working conditions:

  • Store working aliquots at 4°C for up to one week

  • Avoid repeated freeze-thaw cycles as they significantly compromise protein integrity and activity

  • Maintain sterile conditions during handling to prevent microbial contamination

The shelf life is influenced by multiple factors including storage state, buffer composition, storage temperature, and the intrinsic stability of the protein itself .

How can I assess the enzymatic activity of Recombinant Helicobacter pylori Ferrochelatase in experimental settings?

Assessing the enzymatic activity of Recombinant H. pylori Ferrochelatase requires specific methodologies that monitor the conversion of protoporphyrin IX to heme:

• Spectrophotometric assay: This method measures the decrease in fluorescence of protoporphyrin IX as it is converted to heme. The reaction mixture typically contains:

  • Recombinant ferrochelatase (0.1-1 μM)

  • Protoporphyrin IX (0.5-2 μM)

  • Ferrous iron (10-50 μM as ferrous ammonium sulfate)

  • Reducing agent (e.g., 2-mercaptoethanol)

  • Buffer (typically phosphate or Tris-HCl, pH 7.5-8.0)

• HPLC-based quantification: This more sensitive approach directly quantifies the heme product:

  • The reaction is stopped with acid or organic solvent

  • Products are separated by reverse-phase HPLC

  • Heme is detected by absorption at 400-410 nm

• Bioassay approach: Similar to the methodology described for human ferrochelatase, a sensitive bioassay can detect interactions with inhibitors like N-alkylprotoporphyrin IX at concentrations as low as 10⁻⁶ nmol .

When conducting these assays, it's essential to include appropriate controls to account for non-enzymatic iron insertion and potential inhibitory effects from reaction components.

How can Recombinant Helicobacter pylori Ferrochelatase be used in H. pylori vaccine development research?

While Ferrochelatase (hemH) itself is not typically the primary antigen in H. pylori vaccine development, the methodologies and expression systems used for recombinant hemH production share similarities with those used for vaccine antigen candidates. Researchers can leverage these approaches in the following ways:

• Expression system optimization: The baculovirus expression system used for recombinant hemH can be adapted for the production of H. pylori vaccine antigens such as HpaA. The protocols for cultivation, induction, and purification can be modified based on specific antigen requirements .

• Fermentation condition optimization: Similar to the approach used for enhancing HpaA yield, researchers can employ statistical optimization strategies:

  • One-factor-at-a-time analysis to identify significant variables

  • Plackett-Burman factorial experiments to screen key factors

  • Response surface methodology (RSM) and artificial neural network (ANN) models to determine optimal conditions

• Antigenicity and immunogenicity assessment: Methodologies used to validate vaccine candidates can be applied to evaluate potential roles of hemH:

  • Western blot analysis to confirm antigen recognition by antibodies

  • ELISA quantification of serum IgG to assess immune response

  • Animal immunization experiments to evaluate protective efficacy

This research approach has demonstrated significant improvements in yield, with studies showing up to 93.2% increases in recombinant protein production when optimal conditions are identified .

What are the key differences between H. pylori Ferrochelatase and human Ferrochelatase that can be exploited for antimicrobial development?

Structural and functional differences between H. pylori and human ferrochelatases present potential targets for selective inhibition:

These differences can be exploited through:

• Structure-based drug design targeting unique structural features of the H. pylori enzyme

• Development of transition-state analogs specific to the H. pylori catalytic mechanism

• Creation of competitive inhibitors that exploit differences in substrate binding sites

• Design of irreversible inhibitors that selectively modify non-conserved residues in the bacterial enzyme

Understanding these differences requires advanced structural biology techniques including X-ray crystallography, molecular dynamics simulations, and site-directed mutagenesis studies to validate potential drug targets.

How can recombinant ferrochelatase be used as a bioassay system for detecting inhibitors and toxic compounds?

Recombinant ferrochelatase provides a highly sensitive bioassay system for detecting inhibitory compounds, particularly N-alkylprotoporphyrin IX (N-alkylPP). The methodology involves:

• Preparation of the bioassay system:

  • Purified recombinant ferrochelatase is prepared at optimal concentration

  • Reaction components (protoporphyrin IX, ferrous iron, buffer) are standardized

  • Baseline enzyme activity is established under controlled conditions

• Detection of inhibitors:

  • The system can detect N-alkylPP in concentrations as low as 10⁻⁶ nmol

  • Inhibition percentage is calculated by comparing enzyme activity in the presence and absence of test compounds

  • Dose-response curves determine IC₅₀ values for comparative analysis

• Applications to toxicology research:

  • Evaluation of porphyrinogenic xenobiotics that form N-alkylPP after metabolism

  • Assessment of cytochrome P450-mediated bioactivation of compounds

  • Investigation of mechanism-based inactivation pathways

This bioassay approach offers significant advantages over traditional methods:

• Higher sensitivity compared to TLC and UV-visible spectrophotometry

• Reduced sample volume requirements

• Ability to detect inhibitors formed in low-yield in vitro systems

• Potential compatibility with human tissue samples for translational research

What experimental approaches can be used to investigate the role of Ferrochelatase in H. pylori colonization and pathogenesis?

Advanced experimental approaches to investigate ferrochelatase's role in H. pylori pathogenesis include:

• Genetic manipulation strategies:

  • CRISPR-Cas9 genome editing to create hemH knockdown or conditional mutants

  • Complementation studies with wild-type and mutant hemH alleles

  • Site-directed mutagenesis to identify critical catalytic residues

  • Reporter gene fusions to study hemH expression under various conditions

• In vitro infection models:

  • Gastric epithelial cell co-culture systems

  • Polarized cell monolayers to study bacterial adhesion and invasion

  • Microfluidic devices to mimic gastric microenvironments

  • Three-dimensional organoid cultures derived from human gastric tissue

• Animal model approaches:

  • Transgenic mouse models with controlled expression of hemH

  • Germ-free animal models to study H. pylori colonization

  • Competition assays between wild-type and hemH-modified strains

  • In vivo imaging to track bacterial colonization and inflammation

• Omics-based systems biology:

  • Transcriptomics to identify hemH-dependent gene networks

  • Proteomics to characterize the impact of hemH modulation on the bacterial proteome

  • Metabolomics to assess alterations in heme-dependent pathways

  • Host response analysis through immunoprofiling

These multidisciplinary approaches can reveal how ferrochelatase contributes to H. pylori's ability to establish and maintain infection, potentially identifying new therapeutic targets.

What are common challenges in expressing and purifying functional Recombinant Helicobacter pylori Ferrochelatase and how can they be addressed?

Researchers commonly encounter several challenges when working with recombinant ferrochelatase:

Methodological approaches to overcome these challenges:

• Consider using baculovirus expression systems, which have demonstrated success in producing functional H. pylori ferrochelatase with >85% purity .

• Implement a systematic optimization strategy similar to that used for other H. pylori proteins:

  • Apply statistical models (Response Surface Methodology, Artificial Neural Networks) to identify optimal expression conditions

  • Use one-factor-at-a-time approaches to identify critical variables affecting protein yield and activity

• For storage and handling:

  • Add glycerol (5-50%) to prevent freeze-thaw damage

  • Aliquot and store at -20°C/-80°C to maintain stability

  • Avoid repeated freeze-thaw cycles which significantly reduce activity

How can I optimize experimental conditions to study the enzymatic kinetics of Recombinant Helicobacter pylori Ferrochelatase?

Optimizing enzymatic kinetics studies for recombinant H. pylori ferrochelatase requires careful consideration of multiple parameters:

• Buffer optimization:

  • Test multiple buffer systems (HEPES, Tris, phosphate) at pH range 7.0-8.5

  • Evaluate effects of ionic strength (50-200 mM)

  • Assess impact of divalent cations (Mg²⁺, Ca²⁺, Zn²⁺) at various concentrations

  • Include reducing agents (DTT, β-mercaptoethanol) to maintain enzyme in reduced state

• Substrate considerations:

  • Protoporphyrin IX solubility: Use appropriate solvent systems (e.g., DMSO ≤1% final concentration)

  • Iron source: Compare ferrous ammonium sulfate, ferrous ascorbate, and other Fe²⁺ sources

  • Maintain anaerobic conditions to prevent ferrous iron oxidation

• Reaction monitoring:

  • Real-time spectrofluorometric monitoring of protoporphyrin IX consumption

  • HPLC-based product quantification for increased sensitivity

  • Consider stopped-flow techniques for rapid kinetics

• Data analysis optimization:

  • Apply multiple enzyme kinetics models (Michaelis-Menten, Hill equation, etc.)

  • Use global fitting approaches for complex kinetic mechanisms

  • Implement statistical validation of kinetic parameters

• Control experiments:

  • Include heat-inactivated enzyme controls

  • Perform metal-free controls to account for background reactions

  • Test for product inhibition effects

By systematically optimizing these conditions, researchers can obtain reliable kinetic parameters (K_m, k_cat, V_max) that accurately reflect the catalytic properties of H. pylori ferrochelatase.

What approaches can resolve data inconsistencies when comparing different batches of Recombinant Helicobacter pylori Ferrochelatase?

Addressing batch-to-batch variability in recombinant protein studies is crucial for experimental reproducibility and reliable data interpretation:

• Standardized characterization protocol:

  • Implement comprehensive protein characterization for each batch:

    • SDS-PAGE for purity assessment (aim for >85% purity)

    • Mass spectrometry for molecular weight confirmation

    • Circular dichroism for secondary structure verification

    • Activity assays using standardized substrates and conditions

• Reference standards establishment:

  • Create an internal reference standard with well-characterized properties

  • Express and purify a large batch of protein as a reference

  • Aliquot and store under optimal conditions (-80°C with 50% glycerol)

  • Use this standard to normalize data across experiments

• Normalization strategies:

  • Specific activity normalization: Calculate enzyme units per mg of protein

  • Active site titration: Determine the concentration of functionally active enzyme

  • Statistical normalization: Apply correction factors based on reference standards

• Experimental design considerations:

  • Include internal controls in each experiment

  • Perform side-by-side comparisons when possible

  • Use multiple batches to ensure reliability of critical findings

  • Implement factorial experimental designs to account for batch effects

• Data integration approach:

  • Apply meta-analysis techniques to combine data from multiple batches

  • Use statistical models that account for batch as a random effect

  • Consider Bayesian approaches for data integration across experiments

These methodological approaches allow researchers to distinguish true biological effects from technical variability, improving the reliability and reproducibility of research involving recombinant H. pylori ferrochelatase.

How might Recombinant Helicobacter pylori Ferrochelatase contribute to developing new diagnostic methods for H. pylori infection?

Recombinant H. pylori ferrochelatase offers several promising avenues for diagnostic development:

• Serological diagnostics:

  • Development of enzyme-linked immunosorbent assays (ELISAs) using recombinant ferrochelatase as a capture antigen

  • Multiplex serological panels combining ferrochelatase with other H. pylori-specific antigens

  • Lateral flow immunoassays for point-of-care testing

  • Validation studies comparing ferrochelatase-based tests with established diagnostic methods

• Molecular diagnostics:

  • PCR-based detection of hemH gene sequences in clinical samples

  • CRISPR-Cas diagnostic systems targeting hemH

  • Loop-mediated isothermal amplification (LAMP) assays for resource-limited settings

  • Next-generation sequencing approaches to detect multiple H. pylori genes including hemH

• Functional diagnostics:

  • Enzymatic activity-based assays detecting ferrochelatase in biological samples

  • Breath tests based on metabolic pathways involving heme biosynthesis

  • Imaging probes targeting ferrochelatase activity in the gastric environment

• Research applications:

  • Use of recombinant ferrochelatase as a highly sensitive bioassay system (detection limit of 10⁻⁶ nmol)

  • Development of screening methods for compounds that interact with ferrochelatase

  • Investigation of strain-specific variations in ferrochelatase structure and function

These diagnostic approaches must undergo rigorous validation compared to gold standard methods, with careful assessment of sensitivity, specificity, positive predictive value, and negative predictive value in diverse patient populations.

What are the emerging approaches for studying structure-function relationships in H. pylori Ferrochelatase?

Cutting-edge approaches for investigating structure-function relationships in H. pylori ferrochelatase include:

• Advanced structural biology techniques:

  • Cryo-electron microscopy (cryo-EM) for high-resolution structure determination

  • X-ray free-electron laser (XFEL) crystallography for capturing enzymatic intermediates

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to probe protein dynamics

  • Nuclear magnetic resonance (NMR) spectroscopy for solution-state structural analysis

• Computational methodologies:

  • Molecular dynamics simulations to study conformational changes during catalysis

  • Quantum mechanics/molecular mechanics (QM/MM) for modeling the reaction mechanism

  • Deep learning approaches for predicting protein-substrate interactions

  • In silico mutagenesis and virtual screening for inhibitor discovery

• Functional genomics integration:

  • CRISPR-Cas9 scanning mutagenesis to identify essential residues

  • Deep mutational scanning to comprehensively map sequence-function relationships

  • Suppressor mutation analysis to identify functional networks

  • Genomic analysis across H. pylori strains to correlate sequence variations with enzymatic properties

• Single-molecule techniques:

  • Förster resonance energy transfer (FRET) to monitor protein dynamics

  • Atomic force microscopy (AFM) to study protein-protein and protein-substrate interactions

  • Single-molecule enzyme kinetics to characterize reaction intermediates

  • Optical tweezers to investigate mechanical properties of protein folding

These multidisciplinary approaches provide complementary insights into ferrochelatase's structural determinants of function, potentially revealing novel targets for therapeutic intervention.

How can systems biology approaches integrate Ferrochelatase research into broader understanding of H. pylori pathogenesis?

Systems biology offers powerful frameworks to contextualize ferrochelatase within H. pylori's complex pathogenic mechanisms:

• Multi-omics integration:

  • Transcriptomics: RNA-seq analysis of hemH expression under various environmental conditions

  • Proteomics: Quantification of ferrochelatase and interacting proteins in response to host factors

  • Metabolomics: Profiling of heme and related metabolites during infection

  • Integration of these datasets to construct comprehensive models of H. pylori adaptation

• Network biology approaches:

  • Protein-protein interaction networks identifying ferrochelatase binding partners

  • Metabolic flux analysis of heme biosynthesis pathway dynamics

  • Regulatory network reconstruction to identify factors controlling hemH expression

  • Cross-species network comparisons to identify conserved and divergent features

• Host-pathogen interaction modeling:

  • Mathematical modeling of iron acquisition during colonization

  • Agent-based models of bacterial microcolony formation in the gastric environment

  • In silico prediction of host immune responses to bacterial heme metabolism

  • Systems pharmacology approaches to identify multi-target intervention strategies

• Translational systems biology:

  • Integration of clinical metadata with molecular profiles

  • Patient stratification based on bacterial heme metabolism signatures

  • Identification of biomarkers associated with disease progression

  • Prediction of therapeutic responses based on systems-level features

By implementing these approaches, researchers can position ferrochelatase research within a comprehensive understanding of H. pylori pathogenesis, potentially revealing emergent properties and unexpected therapeutic opportunities that would not be apparent from reductionist approaches alone.

How can findings from Recombinant Helicobacter pylori Ferrochelatase research be translated to clinical applications?

The translation of H. pylori ferrochelatase research into clinical applications involves several strategic pathways:

• Therapeutic development:

  • Structure-based design of selective ferrochelatase inhibitors

  • Combination approaches targeting multiple metabolic pathways

  • Repurposing of existing drugs with hemH-modulating activity

  • Development of delivery systems targeting H. pylori's gastric niche

• Diagnostic innovation:

  • Creation of ferrochelatase-based biomarkers for infection detection

  • Development of rapid point-of-care tests utilizing recombinant ferrochelatase

  • Integration with emerging diagnostic platforms (e.g., smartphone-based detection)

  • Pharmacogenomic testing to predict antibiotic resistance based on hemH variations

• Vaccine strategies:

  • Evaluation of ferrochelatase as a potential vaccine antigen

  • Application of optimization methods from recombinant protein production to enhance vaccine manufacturing

  • Development of adjuvant systems compatible with ferrochelatase-based antigens

  • Clinical trial design for ferrochelatase-containing multivalent vaccines

• Precision medicine implementation:

  • Stratification of patients based on H. pylori strain characteristics

  • Tailoring of treatment regimens based on bacterial metabolic profiles

  • Development of companion diagnostics for ferrochelatase-targeting therapeutics

  • Integration with electronic health records for improved clinical decision support

The translation pathway requires interdisciplinary collaboration between basic scientists, clinician-researchers, bioengineers, and pharmaceutical developers to navigate the complex process from laboratory discovery to clinical implementation.

What methodological considerations are important when comparing enzyme kinetics of wild-type versus mutant forms of Recombinant Helicobacter pylori Ferrochelatase?

• Protein quality standardization:

  • Express and purify all protein variants using identical protocols

  • Verify structural integrity through circular dichroism or thermal stability assays

  • Confirm purity levels (>85% by SDS-PAGE) for all variants

  • Quantify protein concentration using multiple methods (Bradford, BCA, A280)

• Experimental design optimization:

  • Conduct kinetic assays under identical conditions (buffer, temperature, pH)

  • Include both internal and external controls in each experimental run

  • Perform experiments with multiple protein preparations to account for batch effects

  • Use factorial design to systematically assess interaction effects

• Comprehensive kinetic analysis:

  • Determine full kinetic parameters (K_m, k_cat, k_cat/K_m) for all substrates

  • Investigate potential changes in reaction mechanism

  • Assess product inhibition effects

  • Evaluate pH-dependence profiles to identify changes in ionizable groups

• Advanced analytical approaches:

  • Apply global data fitting to complex kinetic models

  • Use statistical methods that account for error propagation

  • Conduct sensitivity analysis to identify robust differences

  • Consider Bayesian approaches for parameter estimation

• Structural-functional correlation:

  • Correlate kinetic changes with specific structural alterations

  • Use molecular dynamics simulations to interpret experimental findings

  • Apply transition state theory to understand catalytic efficiency differences

  • Consider evolutionary conservation of mutated residues

These methodological considerations ensure that observed differences between wild-type and mutant enzymes reflect genuine biological phenomena rather than experimental artifacts.

What are the critical factors for scaling up production of Recombinant Helicobacter pylori Ferrochelatase for research purposes?

Scaling up production of recombinant H. pylori ferrochelatase requires systematic optimization of multiple parameters:

• Expression system optimization:

  • Evaluate different expression hosts (bacterial, insect, mammalian) for optimal yield and activity

  • Compare baculovirus expression systems with E. coli-based systems

  • Optimize codon usage for the selected expression host

  • Engineer expression constructs with appropriate fusion tags and cleavage sites

• Fermentation process development:

  • Implement statistical optimization methodologies like Response Surface Methodology (RSM) and Artificial Neural Network (ANN) models

  • Identify critical medium components (glucose, yeast extract, nitrogen sources) affecting yield

  • Optimize induction parameters (timing, temperature, inducer concentration)

  • Scale up from shake flasks to controlled bioreactors with monitoring of key parameters

• Purification process optimization:

  • Develop robust chromatography protocols ensuring >85% purity

  • Implement tangential flow filtration for efficient volume reduction

  • Optimize buffer conditions to maintain protein stability

  • Consider automated purification systems for reproducibility

• Quality control implementation:

  • Establish specifications for purity, activity, and stability

  • Implement in-process controls at critical steps

  • Develop validated analytical methods for product characterization

  • Institute stability testing protocols under various storage conditions