Recombinant Mustela lutreola Hemoglobin subunit beta (HBB)

What is the molecular structure and characteristics of Mustela lutreola HBB?

Mustela lutreola HBB is a 146-amino acid protein belonging to the globin family with a molecular mass of approximately 15.9 kDa . The primary structure is characterized by the following amino acid sequence:

VHLTAEEKAAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFDSFGDLSSPDAVMGNPKVKAHGKKVLNSFSEGLKNLDNLKGTFAKLSELHCDKLHVDPENFKLLGNVLVCVLAHHFGKEFTPQVQAAYQKVVAGVATALAHKYH

Like other beta-globin proteins, it functions primarily in oxygen transport from the lungs to peripheral tissues. The tertiary structure likely contains the characteristic globin fold with alpha-helical segments surrounding a hydrophobic core that houses the heme group. While no specific crystallographic data was presented in the search results for Mustela lutreola HBB, research approaches typically involve comparative structural analysis with other mammalian beta-globins to identify conserved functional domains.

What expression systems are most effective for producing recombinant Mustela lutreola HBB?

Mammalian expression systems appear to be effective for producing functional recombinant hemoglobin subunits. For instance, transient mammalian gene expression has been successfully used for generating recombinant haptoglobin variants that can bind to hemoglobin . This suggests a similar approach could be adapted for Mustela lutreola HBB.

The methodology would typically involve:

• Gene synthesis or cloning of the Mustela lutreola HBB gene

• Insertion into a mammalian expression vector with appropriate promoter and selection markers

• Transfection into a mammalian cell line such as Expi293F cells as demonstrated for other hemoglobin-binding proteins

• Protein purification using affinity chromatography (e.g., with His-tag) followed by size exclusion chromatography

For purification specifically, a method demonstrated for similar proteins involves:

• Initial capture using HisTrap excel columns

• Buffer exchange into PBS using desalting columns

• Preparative separation of aggregate and size species using size exclusion chromatography

This approach allows for proper folding and potential post-translational modifications that may be important for the protein's function.

How can researchers analyze mutations in the HBB gene across species including Mustela lutreola?

Researchers can employ computational approaches similar to those used for human HBB mutation analysis. The SNPEFF tool can be used to align sequencing data with reference genomes . For a comprehensive analysis, researchers should:

• Collect DNA samples from multiple Mustela lutreola individuals

• Perform whole genome or targeted sequencing of the HBB locus

• Align sequences with a reference genome using tools like SNPEFF

• Visualize variations using the Integrative Genomics Viewer (IGV)

• Classify mutations based on type (synonymous, missense, frameshift, etc.)

• Assess potential pathogenicity using multiple prediction tools

To predict the functional consequences of identified mutations, researchers should employ multiple prediction tools as demonstrated in human HBB studies:

Using multiple predictors improves accuracy, as demonstrated in human HBB mutation studies that identified pathogenic variants with a high degree of confidence .

What methodologies can be employed for comparative oxygen-binding studies between recombinant and native Mustela lutreola HBB?

To conduct rigorous comparative oxygen-binding studies between recombinant and native Mustela lutreola HBB, researchers should implement a multi-technique approach:

• Oxygen Equilibrium Curve (OEC) Analysis:

  • Use a spectrophotometric method with a tonometer to measure oxygen saturation at varying oxygen partial pressures

  • Calculate P50 (oxygen tension at 50% saturation) and Hill coefficient (n) to quantify cooperativity

  • Compare these parameters between recombinant and native proteins under identical buffer conditions

• Stopped-Flow Kinetic Measurements:

  • Employ rapid mixing techniques to measure the rates of oxygen association (kon) and dissociation (koff)

  • Calculate the ratio koff/kon to derive the equilibrium constant (KD)

  • Measure these parameters at different temperatures to determine thermodynamic parameters (ΔH, ΔS)

• Effect of Allosteric Modulators:

  • Test the influence of physiological modulators (pH, 2,3-BPG, chloride ions) on oxygen binding

  • Generate comparative data tables showing the shift in P50 values in response to these modulators

Discrepancies between recombinant and native protein binding kinetics may indicate differences in post-translational modifications or folding patterns. Such differences would require further investigation through mass spectrometry and circular dichroism analyses to identify structural variations.

How can CRISPR/Cas9 techniques be optimized for studying or modifying the HBB gene in Mustela lutreola models?

CRISPR/Cas9 methodology can be adapted for Mustela lutreola HBB gene modification based on approaches developed for human HBB gene therapy. The following protocol would be appropriate:

• sgRNA Design and Validation:

  • Design multiple sgRNAs targeting conserved regions of the Mustela lutreola HBB gene

  • Evaluate off-target effects using computational prediction tools

  • Test sgRNA efficiency using in vitro cleavage assays with synthesized target DNA

• Homology-Directed Repair (HDR) Template Design:

  • Develop HDR templates with 800-1000bp homology arms flanking the target region

  • Include desired modifications (e.g., fluorescent tags, specific mutations)

  • Consider using CRISPR/Cas9 to enhance HDR efficiency as demonstrated in human HBB gene therapy approaches

• Delivery Methods:

  • For cell culture: Use nucleofection or lipid-based transfection of ribonucleoprotein complexes

  • For in vivo: Consider AAV or lentiviral vectors for delivery to specific tissues

• Validation of Editing Efficiency:

  • Perform targeted deep sequencing to quantify editing rates

  • Use restriction fragment length polymorphism (RFLP) analysis for rapid screening

  • Employ droplet digital PCR for precise quantification of editing events

This approach would allow for precise modification of the Mustela lutreola HBB gene, enabling functional studies or development of disease models. The universal strategy developed for repairing various HBB mutations in human stem cells could be adapted for Mustela lutreola models, particularly for comparative studies of hemoglobinopathies .

What are the optimal protocols for purification and structural characterization of recombinant Mustela lutreola HBB?

A comprehensive purification and structural characterization protocol for recombinant Mustela lutreola HBB should include:

• Multi-step Purification Process:

  • Initial capture using immobilized metal affinity chromatography (IMAC) with His-tagged protein

  • Intermediate purification using ion exchange chromatography to separate charge variants

  • Polishing step using size exclusion chromatography in PBS buffer

  • Quality assessment by SDS-PAGE and Western blotting

• Structural Characterization:

  • Circular dichroism (CD) spectroscopy to analyze secondary structure content

  • Differential scanning calorimetry (DSC) to determine thermal stability

  • Nuclear magnetic resonance (NMR) for solution structure determination

  • X-ray crystallography for high-resolution structural analysis

• Functional Validation:

  • UV-visible spectroscopy to confirm proper heme incorporation

  • Oxygen binding assays to verify functional activity

  • Comparative analysis with native protein to ensure recombinant protein faithfully represents the natural form

The specific buffer conditions for optimal stability should be determined empirically, but starting conditions could include PBS (pH 7.4) with potential additives such as glycerol (10%) to enhance stability during storage . This methodological approach ensures the production of high-quality protein suitable for downstream structural and functional studies.

How can researchers investigate the evolutionary significance of Mustela lutreola HBB compared to other mustelid species?

To investigate the evolutionary significance of Mustela lutreola HBB, researchers should implement a comprehensive phylogenetic and functional comparative analysis:

• Sequence Alignment and Phylogenetic Analysis:

  • Collect HBB sequences from multiple mustelid species and other related mammals

  • Perform multiple sequence alignment using tools like MUSCLE or CLUSTAL

  • Construct phylogenetic trees using maximum likelihood or Bayesian methods

  • Calculate sequence identity and similarity percentages across species

• Selection Pressure Analysis:

  • Calculate the ratio of non-synonymous to synonymous substitution rates (dN/dS)

  • Identify sites under positive, neutral, or purifying selection

  • Map these sites onto the three-dimensional structure to assess functional significance

• Functional Divergence Assessment:

  • Express recombinant HBB from multiple mustelid species

  • Compare oxygen binding properties, cooperativity, and responses to allosteric effectors

  • Correlate functional differences with habitat and physiological adaptations

• Ecological Correlation Analysis:

  • Gather data on habitat preferences, diving behavior, and altitude ranges for each species

  • Create a correlation matrix between HBB sequence variations and ecological parameters

  • Test for statistically significant associations between specific amino acid changes and ecological adaptations

This approach would provide insights into how natural selection has shaped the evolution of HBB in Mustela lutreola and related species, potentially revealing molecular adaptations that correlate with specific ecological niches or physiological demands.

What methodologies can be employed to study the interaction between recombinant Mustela lutreola HBB and potential binding partners?

To thoroughly investigate protein-protein interactions involving recombinant Mustela lutreola HBB, researchers should employ multiple complementary techniques:

• Surface Plasmon Resonance (SPR):

  • Immobilize purified recombinant HBB on a sensor chip

  • Flow potential binding partners over the surface at varying concentrations

  • Determine association (kon) and dissociation (koff) rate constants

  • Calculate equilibrium dissociation constants (KD) for each interaction

• Isothermal Titration Calorimetry (ITC):

  • Measure heat changes upon binding to determine:

    • Binding stoichiometry (n)

    • Binding affinity (KD)

    • Enthalpy changes (ΔH)

    • Calculate entropy contributions (ΔS)

• Co-Immunoprecipitation and Pull-down Assays:

  • Use tagged recombinant HBB to pull down interacting partners from cell lysates

  • Identify novel binding partners through mass spectrometry analysis

  • Confirm interactions through reciprocal pull-downs

• Biolayer Interferometry (BLI):

  • Immobilize HBB on biosensors and measure real-time binding kinetics

  • Screen multiple potential interacting proteins in parallel

  • Validate binding specificity through competition assays

• Molecular Docking and Simulation:

  • Generate structural models of Mustela lutreola HBB

  • Perform in silico docking with potential binding partners

  • Validate predicted interactions experimentally

For interactions with heme scavenger proteins like haptoglobin, methodological approaches similar to those described for human hemoglobin could be adapted . This would involve assessing complex formation through size exclusion chromatography and validating functional consequences such as antioxidant properties and nitric oxide sparing capacity.

How can recombinant Mustela lutreola HBB serve as a model for studying hemoglobinopathies?

Recombinant Mustela lutreola HBB can serve as a valuable comparative model for human hemoglobinopathies through the following methodological approaches:

• Engineered Mutation Studies:

  • Introduce mutations corresponding to human hemoglobinopathies (e.g., sickle cell mutation) into the Mustela lutreola HBB gene

  • Express and purify these mutant proteins

  • Compare structural and functional consequences with human mutant HBB

  • Assess how differences in protein context affect the impact of pathogenic mutations

• Comparative Analysis of Mutation Tolerance:

  • Create a mutation tolerance map for Mustela lutreola HBB

  • Compare with human HBB tolerance maps

  • Identify regions with differential sensitivity to mutations

  • Investigate structural or functional basis for these differences

• Cross-Species Complementation Studies:

  • Test if Mustela lutreola HBB can functionally substitute for human HBB in cellular systems

  • Evaluate the potential of chimeric hemoglobins containing elements from both species

  • Assess the impact on oxygen binding, stability, and interactions with other proteins

Such studies could provide insights into the molecular basis of hemoglobinopathies and potentially identify novel therapeutic strategies. The comparative approach leverages evolutionary differences to understand fundamental structure-function relationships in beta-globin proteins.

What are the challenges and solutions in designing CRISPR/Cas9-based therapeutic approaches for HBB gene correction based on animal models?

Developing CRISPR/Cas9 therapeutic approaches using insights from animal models like Mustela lutreola presents several challenges that require methodological solutions:

• Species-Specific Differences in DNA Repair Mechanisms:

  • Challenge: Efficiency of homology-directed repair (HDR) varies across species

  • Solution: Comparative analysis of repair outcomes in different cell types; optimization of template design for each species; use of HDR enhancers

• Delivery Methods for In Vivo Application:

  • Challenge: Efficient delivery to target tissues varies by species

  • Solution: Systematic testing of viral and non-viral delivery systems; species-specific optimization of vector tropism; development of tissue-targeted delivery approaches

• Off-Target Effects and Specificity:

  • Challenge: Off-target profiles differ between species due to genome differences

  • Solution: Whole-genome sequencing to identify species-specific off-target sites; use of high-fidelity Cas9 variants; thorough validation across models

• Immune Responses to CRISPR Components:

  • Challenge: Species-specific immune reactions to Cas9 and delivery vehicles

  • Solution: Transient expression systems; immunomodulation strategies; development of less immunogenic delivery methods

The universal approach described for correcting various human HBB mutations offers a methodological framework that could be adapted to different species, potentially allowing for comparative studies of correction efficiency and optimization strategies . Such cross-species analyses would strengthen the translational potential of CRISPR/Cas9 therapies for hemoglobinopathies.

How can structural studies of Mustela lutreola HBB inform protein engineering approaches for oxygen carriers?

Structural studies of Mustela lutreola HBB can provide valuable insights for protein engineering of oxygen carriers through the following methodological approach:

• Comparative Structural Analysis:

  • Determine the crystal structure of Mustela lutreola HBB at high resolution

  • Compare with human HBB and other mammalian hemoglobins

  • Identify unique structural features that may confer advantageous properties

  • Map sequence differences onto structural models to understand their functional significance

• Structure-Function Relationship Determination:

  • Create chimeric proteins incorporating domains from Mustela lutreola and other species

  • Assess how specific structural elements contribute to:

    • Oxygen affinity and cooperativity

    • Stability under various conditions

    • Resistance to oxidation

    • Interaction with regulatory molecules

• Rational Design of Improved Oxygen Carriers:

  • Use insights from Mustela lutreola HBB to design hemoglobin variants with:

    • Optimized oxygen binding properties for specific applications

    • Enhanced stability for longer circulation times

    • Reduced nitric oxide scavenging to minimize vasoconstriction

    • Controlled autooxidation rates to prevent hemoglobin-induced oxidative stress

• Validation in Physiologically Relevant Systems:

  • Test engineered variants in red blood cell substitutes

  • Assess oxygen delivery in tissue perfusion models

  • Evaluate performance under various physiological stresses

This approach leverages evolutionary adaptations that may be present in Mustela lutreola HBB to inform the development of next-generation hemoglobin-based oxygen carriers with improved properties for therapeutic applications.