Recombinant Theromyzon tessulatum Ovohemerythrin

What is Theromyzon tessulatum Ovohemerythrin and how does it relate to other proteins isolated from this leech species?

Theromyzon tessulatum Ovohemerythrin is an oxygen-binding protein found in the rhynchobdellid leech Theromyzon tessulatum. While specific literature on this protein is limited, it belongs to the hemerythrin family of non-heme iron proteins that function as oxygen carriers in some invertebrates. Unlike hemoglobin, hemerythrins use a di-iron center for oxygen binding rather than a heme group. Theromyzon tessulatum is known to produce several other well-characterized proteins, including a 32 kDa aspartyl protease with renin-like activity and thrombin inhibitors such as theromin . The study of Ovohemerythrin should be considered in the context of the leech's broader proteome, where various proteins have been identified through techniques such as gel permeation chromatography, affinity column separation, and HPLC purification .

What analytical techniques are most effective for initial characterization of recombinant Theromyzon tessulatum Ovohemerythrin?

For initial characterization, a multi-method approach is recommended:

• Primary sequence determination: Combine reduction and s-β-pyridylethylation, Edman degradation, enzymatic digestion (typically with trypsin), and mass spectrometry (particularly MALDI-TOF MS) .

• Structural analysis: Employ circular dichroism spectroscopy for secondary structure assessment, X-ray crystallography for high-resolution structure, and NMR for solution-state conformational studies.

• Functional characterization: Oxygen-binding assays using spectrophotometric methods to monitor changes in absorption spectra upon oxygenation/deoxygenation.

• Purity assessment: SDS-PAGE, native PAGE, and size exclusion chromatography to evaluate homogeneity and oligomeric state.

A systematic characterization approach similar to that used for theromin isolation would involve gel permeation chromatography, ion exchange chromatography, and reverse-phase HPLC steps .

What expression systems are optimal for producing recombinant Theromyzon tessulatum Ovohemerythrin?

The optimal expression system selection should consider:

For initial studies, an E. coli system with a fusion tag (such as His6, GST, or MBP) is recommended, as similar approaches have been successful for other leech-derived proteins. If proper folding becomes problematic, consider co-expression with chaperones or switching to insect cell systems. Expression should be optimized through systematic variation of induction conditions, similar to methodologies outlined in proteomics studies for other organisms .

What purification strategy provides the highest yield and purity of recombinant Theromyzon tessulatum Ovohemerythrin?

A multi-step purification strategy is recommended based on approaches used for other leech proteins:

• Initial capture: Affinity chromatography (if expressed with a tag) or ion exchange chromatography based on predicted isoelectric point.

• Intermediate purification: Gel permeation chromatography to separate by molecular size.

• Polishing step: Reverse-phase HPLC for final purification to homogeneity.

For optimal results, follow a three-step purification protocol similar to that used for the renin-like enzyme from Theromyzon tessulatum, which included gel permeation chromatography, affinity column separation, and reversed-phase HPLC . This approach has demonstrated effectiveness in purifying leech proteins to homogeneity.

Monitoring purity throughout the process using SDS-PAGE and activity assays is essential, with expected final purity exceeding 95% as determined by densitometric analysis of stained gels.

How can sequence homology analysis help predict functional domains in Theromyzon tessulatum Ovohemerythrin?

Sequence homology analysis serves as a powerful predictive tool for identifying functional domains in Theromyzon tessulatum Ovohemerythrin. The methodological approach should include:

• Sequence alignment with other hemerythrins: Use multiple sequence alignment tools (MUSCLE, CLUSTAL Omega) to identify conserved regions that may correspond to oxygen-binding sites or structural motifs.

• Domain prediction: Employ specialized databases like PROSITE, Pfam, and InterPro to identify characteristic domains and motifs.

• Evolutionary conservation mapping: Analyze patterns of sequence conservation across species to identify functionally important residues.

• Structure prediction: Utilize homology modeling based on known hemerythrin structures to predict the three-dimensional arrangement of functional domains.

When analyzing Theromyzon tessulatum proteins, it's instructive to note the approach used for the renin-like enzyme, where sequence analysis revealed 26.5-35.5% identity with mammalian counterparts in the N-terminal region, and a highly conserved region (80% homology) containing the catalytic aspartyl residue . Similar analysis of theromin showed no significant sequence homology with other thrombin inhibitors, highlighting the importance of comprehensive analysis beyond simple homology searches .

What spectroscopic methods are most informative for studying the metal-binding sites in Theromyzon tessulatum Ovohemerythrin?

For detailed characterization of metal-binding sites in Ovohemerythrin, employ a combination of complementary spectroscopic techniques:

• UV-Visible spectroscopy: Monitor characteristic absorption bands associated with metal-coordination environments and changes upon oxygen binding.

• Electron Paramagnetic Resonance (EPR): Detect paramagnetic species and characterize the oxidation states of iron centers in different functional states.

• Mössbauer spectroscopy: Provide detailed information about the electronic environment, oxidation state, and magnetic properties of iron atoms.

• X-ray Absorption Spectroscopy (XAS): Determine metal-ligand distances and coordination geometries with high precision.

• Resonance Raman spectroscopy: Characterize vibrational modes associated with metal-oxygen bonds.

Each technique provides unique information about the di-iron center. Data integration from multiple methods is essential for a complete understanding of the metal-binding site structure and function in different oxidation and ligation states.

How should researchers design experiments to study oxygen-binding properties of recombinant Theromyzon tessulatum Ovohemerythrin?

A systematic experimental design approach should include:

• Equilibrium binding studies: Use spectrophotometric methods to determine oxygen affinity (P50) and cooperativity (Hill coefficient) under varying conditions (pH, temperature, salt concentration).

• Kinetic measurements: Employ stopped-flow techniques to measure rates of oxygen association and dissociation.

• Environmental variables: Systematically test the effects of pH (5.0-9.0), temperature (4-37°C), and ionic strength on oxygen binding properties.

• Comparative analysis: Include other oxygen-binding proteins as controls to benchmark results.

• Mutagenesis experiments: Create strategic mutations in putative oxygen-binding residues to confirm their functional role.

Experimental design should adhere to principles outlined in "The Design of Animal Experiments," including proper randomization, adequate sample size determination, and appropriate controls to ensure statistical validity and minimize animal use . For protein-based experiments, replicate measurements (minimum n=3) are essential, with statistical analysis using ANOVA or similar methods to assess significance.

What control experiments are essential when evaluating the functional properties of recombinant Theromyzon tessulatum Ovohemerythrin?

A comprehensive control strategy should include:

• Negative controls:

  • Buffer-only conditions to establish baseline measurements

  • Heat-denatured protein to confirm specificity of functional assays

  • Non-functional mutants with altered metal-binding sites

• Positive controls:

  • Known oxygen-binding proteins (hemoglobin, myoglobin) for benchmark comparisons

  • Native (non-recombinant) hemerythrin if available

• Specificity controls:

  • Testing with other gaseous ligands (CO, NO) to assess binding specificity

  • Competitive binding assays with known inhibitors

• Technical controls:

  • Multiple protein batches to account for preparation variability

  • Concentration gradients to ensure linearity of response

  • Time-course measurements to establish equilibrium conditions

Control experiments should be incorporated into the experimental design as outlined in established laboratory animal research guidelines, ensuring that all variables except the one being tested are held constant . This methodological rigor helps distinguish true effects from artifacts and ensures reproducibility.

What approaches can address protein insolubility issues when expressing recombinant Theromyzon tessulatum Ovohemerythrin?

When encountering insolubility challenges with recombinant Ovohemerythrin, implement this systematic troubleshooting framework:

• Expression conditions optimization:

  • Reduce expression temperature (16-25°C)

  • Decrease inducer concentration

  • Use enriched media formulations

  • Extend expression time with lower induction levels

• Construct modifications:

  • Utilize solubility-enhancing fusion partners (MBP, SUMO, TRX)

  • Remove flexible or hydrophobic regions predicted to cause aggregation

  • Codon-optimize the sequence for the expression host

• Buffer optimization:

  • Screen various pH conditions (typically pH 6.0-8.5)

  • Test different salt concentrations (100-500 mM)

  • Add stabilizing agents (glycerol 5-15%, low concentrations of non-ionic detergents)

  • Include metal ions that might stabilize the native fold

• Co-expression strategies:

  • Co-express with molecular chaperones (GroEL/ES, DnaK/J)

  • Include iron transport proteins to ensure proper metallation

Implementing a filter-assisted sample protocol similar to that described for proteomic studies can improve protein recovery and solubility during purification processes . If inclusion bodies form despite these measures, develop a refolding protocol using gradual dialysis with decreasing denaturant concentrations.

How can researchers address data inconsistencies in oxygen-binding measurements of Theromyzon tessulatum Ovohemerythrin?

When confronted with inconsistent oxygen-binding data, employ this methodological approach:

• Technical validation:

  • Verify instrument calibration and performance using standard references

  • Ensure consistent protein concentration determination methods

  • Standardize sample handling procedures

• Sample quality assessment:

  • Check protein purity by multiple methods (SDS-PAGE, mass spectrometry)

  • Verify correct metallation using ICP-MS or colorimetric iron assays

  • Assess protein stability over time using activity measurements

• Experimental variables control:

  • Standardize buffer preparation methods

  • Control temperature precisely during measurements

  • Eliminate oxygen contamination in deoxygenated samples

• Statistical approach:

  • Increase technical replicates (minimum n=5)

  • Use statistical methods that account for outliers

  • Consider hierarchical experimental designs to separate batch effects

For data analysis, implement approaches similar to those used in proteomic studies, where replicate variation is systematically accounted for when identifying significantly regulated proteins . Creating a standardized operating procedure with clearly defined quality control checkpoints will improve consistency across experiments.

How can activity-based protein profiling be applied to study Theromyzon tessulatum Ovohemerythrin function in complex biological samples?

Activity-based protein profiling (ABPP) represents a powerful approach for studying Ovohemerythrin function within complex biological contexts:

• Probe design strategy:

  • Develop oxygen-mimetic probes containing reactive groups that bind to the di-iron center

  • Incorporate reporter tags (fluorescent, biotin) for detection and enrichment

  • Design competitive probes to distinguish specific from non-specific interactions

• Application methodology:

  • Apply probes to intact cells, tissue homogenates, or purified protein

  • Optimize probe concentration, incubation time, and reaction conditions

  • Use gel-based analysis or mass spectrometry for detection

• Data analysis framework:

  • Quantify labeling efficiency under different conditions

  • Compare active site accessibility in different protein states

  • Correlate activity profiles with physiological changes

This approach can determine the activity state of proteins in whole cell proteomes and reveal that enzyme activity can change in response to different conditions independently of expression levels . For Ovohemerythrin, ABPP could provide insights into oxygen-binding capacity under various physiological conditions and identify potential regulatory mechanisms.

What mass spectrometry approaches are most effective for characterizing post-translational modifications in recombinant Theromyzon tessulatum Ovohemerythrin?

A comprehensive mass spectrometry strategy for PTM characterization should include:

• Sample preparation optimization:

  • Multiple proteolytic digestion strategies (trypsin, chymotrypsin, Glu-C)

  • Enrichment techniques for specific modifications (TiO2 for phosphorylation, lectin affinity for glycosylation)

  • Reduction and alkylation to preserve cysteine modifications

• MS/MS analysis techniques:

  • Employ combined approaches of data-dependent acquisition (DDA) and data-independent acquisition (SWATH) for comprehensive coverage

  • Use multiple fragmentation methods (CID, HCD, ETD) for different modification types

  • Implement targeted approaches for quantification of specific modified peptides

• Data analysis workflow:

  • Search against multiple protein databases with variable modification parameters

  • Validate identifications using site-determining ions and retention time prediction

  • Quantify modification stoichiometry using label-free or isotope labeling approaches

This multi-faceted approach allows for comprehensive characterization of PTMs while minimizing false-positive identifications. The combination of DDA and SWATH methodologies has proven effective for proteomic analysis, providing complementary data that enhances coverage and quantification accuracy .

How does Theromyzon tessulatum Ovohemerythrin compare structurally and functionally to other proteins isolated from this leech species?

A systematic comparative analysis reveals important distinctions and relationships:

While specific structural data on Ovohemerythrin is limited, the purification strategies employed for other T. tessulatum proteins provide a valuable methodological framework. The isolation techniques used for theromin (gel permeation and anion exchange chromatography followed by reverse-phase HPLC) represent a proven approach that could be adapted for Ovohemerythrin purification . The detailed sequence analysis methodology employed for the renin-like enzyme, which identified regions of homology with mammalian counterparts, offers a template for comparative sequence analysis of Ovohemerythrin .

What can the study of Theromyzon tessulatum proteins teach us about experimental design for recombinant protein research?

The study of various T. tessulatum proteins yields valuable lessons for experimental design:

• Purification strategy selection:

  • Multi-step approaches combining different principles (size separation, affinity, hydrophobicity) consistently yield high purity

  • The three-step purification protocol (gel permeation, affinity separation, HPLC) used for the renin-like enzyme provides a proven template

• Sequence analysis methodology:

  • Comprehensive approach combining multiple techniques (Edman degradation, enzymatic digestion, mass spectrometry) provides more reliable sequence determination

  • Cross-validation using different approaches enhances confidence in structural assignments

• Functional characterization framework:

  • Activity-based assays provide more physiologically relevant data than simple binding studies

  • Context-dependent activity changes highlight the importance of testing under various conditions

• Experimental design principles:

  • The importance of randomized controlled experimental designs

  • Inclusion of both sexes in experimental designs using factorial approaches

  • Careful selection of experimental animals and appropriate sample sizes

These methodological insights from T. tessulatum protein research directly support principles outlined in laboratory research guidelines, emphasizing that well-designed experiments minimize animal use while maximizing scientific validity .

What techniques can be used to study the effect of temperature on Theromyzon tessulatum Ovohemerythrin activity and stability?

A comprehensive temperature-effect study should employ these methodological approaches:

• Thermal stability analysis:

  • Differential scanning calorimetry (DSC) to determine melting temperature (Tm)

  • Circular dichroism with temperature ramping to monitor secondary structure changes

  • Intrinsic fluorescence spectroscopy to track tertiary structure alterations

  • Thermal shift assays using environment-sensitive dyes

• Functional activity assessment:

  • Oxygen-binding measurements at different temperatures (typically 4-40°C)

  • Stopped-flow kinetics at varying temperatures to calculate activation energies

  • Spectroscopic monitoring of conformational changes associated with oxygen binding

• Long-term stability studies:

  • Activity retention after incubation at different temperatures

  • Analysis of aggregation propensity using dynamic light scattering

  • Monitoring of metal retention using ICP-MS or colorimetric assays

Similar temperature-effect studies on enzyme activity have revealed important insights about protein function, showing that enzyme activity can significantly change in response to temperature independently of expression levels . For Ovohemerythrin, these studies could reveal temperature-dependent conformational changes that affect oxygen affinity and release kinetics.

How can site-directed mutagenesis be used to investigate structure-function relationships in Theromyzon tessulatum Ovohemerythrin?

A strategic site-directed mutagenesis approach should include:

• Target selection methodology:

  • Identify conserved residues through multiple sequence alignment with other hemerythrins

  • Focus on putative metal-coordinating residues (typically histidines)

  • Target residues in the predicted oxygen-binding pocket

  • Investigate residues at subunit interfaces if oligomeric

• Mutation design principles:

  • Conservative substitutions to probe specific interactions (e.g., His→Asn)

  • Charge reversals to disrupt electrostatic networks

  • Alanine scanning of specific regions to identify essential residues

  • Introduction of reporter residues (e.g., Cys, Trp) for spectroscopic studies

• Functional characterization protocol:

  • Compare oxygen-binding parameters (P50, Hill coefficient) between wild-type and mutants

  • Analyze structural stability using thermal denaturation

  • Assess metal-binding capacity through spectroscopic methods

  • Determine oligomeric state changes using analytical ultracentrifugation

This systematic mutagenesis approach allows for detailed mapping of structure-function relationships, similar to methodologies that have been successfully applied to other metalloproteins. The results can be integrated with structural data to develop a comprehensive model of how Ovohemerythrin's structure enables its oxygen-binding function.