Recombinant Sorex hosonoi Cytochrome b (MT-CYB)

What is Recombinant Sorex hosonoi Cytochrome b and what are its primary structural characteristics?

Recombinant Sorex hosonoi Cytochrome b (MT-CYB) is a partial recombinant protein derived from the mitochondrial genome of the Azumi shrew (Sorex hosonoi), produced through heterologous expression systems . This protein is a critical component of the electron transport chain's Complex III, functioning as Complex III subunit 3 (also known as Ubiquinol-cytochrome-c reductase complex cytochrome b subunit) . The protein is available from commercial sources in both yeast-derived and mammalian cell-derived forms, each offering >85% purity as determined by SDS-PAGE analysis . It's identified in the UniProt database under accession number O21414, which provides researchers with access to its known sequence characteristics and functional annotations .

How does Cytochrome b from Sorex hosonoi differ from other mammalian Cytochrome b proteins in terms of sequence conservation?

Cytochrome b is generally well-conserved across mammalian species due to its essential role in energy metabolism, but species-specific variations exist, particularly in regions not critical for electron transport function. While the search results don't provide specific sequence comparisons for Sorex hosonoi cytochrome b versus other mammals, research on related shrew species like Sorex araneus has revealed significant nucleotide sequence variations . In S. araneus, a 572 bp region of the cytochrome b gene showed multiple haplotypes with a distinct star-like network structure, with approximately 36% of individuals sharing a central haplotype . These variations have proven valuable for studying population structure and post-glacial colonization patterns. When designing experiments, researchers should consider that while the functional domains of cytochrome b are highly conserved, species-specific variations may affect antibody recognition, protein-protein interactions, and potentially drug binding characteristics .

What are the recommended reconstitution protocols for Recombinant Sorex hosonoi Cytochrome b to maintain optimal activity?

For optimal reconstitution of lyophilized Recombinant Sorex hosonoi Cytochrome b, follow this methodological approach:

• Centrifuge the vial briefly to ensure all material is at the bottom of the container before opening .

• Reconstitute the protein in deionized sterile water to reach a concentration between 0.1-1.0 mg/mL .

• Add glycerol to a final concentration of 5-50% (with 50% being the manufacturer's default recommendation) to enhance stability during storage .

• Aliquot the reconstituted protein into smaller volumes to avoid repeated freeze-thaw cycles, which can significantly compromise protein integrity and activity .

• Store working aliquots at 4°C for short-term use (up to one week) and store remaining aliquots at -20°C/-80°C for long-term storage .

This reconstitution protocol is designed to maintain the structural integrity and functional activity of the protein, which is crucial for downstream applications such as enzymatic assays, antibody production, and protein-protein interaction studies.

How can Recombinant Sorex hosonoi Cytochrome b be utilized in evolutionary studies of shrews and related mammals?

Recombinant Sorex hosonoi Cytochrome b can serve as a valuable molecular tool in evolutionary studies through several methodological approaches:

• Phylogenetic Analysis: The protein or its encoding sequence can be used as a reference standard when constructing phylogenetic trees to understand evolutionary relationships among Sorex species and other insectivores . Research on Sorex araneus has already demonstrated that cytochrome b sequences can reveal population structuring and colonization patterns .

• Haplotype Network Construction: The gene can be used to generate median haplotype networks similar to those observed in S. araneus, which showed a pronounced star-like structure indicative of population expansion after bottleneck events .

• Post-glacial Colonization Studies: Cytochrome b sequence data can inform hypotheses about historical population movements, as demonstrated in research showing how S. araneus likely colonized post-glacial territories through specific matrilines .

• Chromosome Race Analysis: When combined with karyotype data, cytochrome b variation can help understand the relationship between chromosomal evolution and genetic divergence in shrews, which exhibit significant chromosomal polymorphism .

• Comparative Molecular Evolution: By comparing recombinant Sorex hosonoi cytochrome b with that of other species, researchers can investigate rates of molecular evolution and selective pressures on mitochondrial genes across different mammalian lineages.

What are the optimal storage conditions for maximizing the shelf life of Recombinant Sorex hosonoi Cytochrome b?

The stability and shelf life of Recombinant Sorex hosonoi Cytochrome b depend significantly on storage conditions, formulation, and handling practices. Optimal storage follows these evidence-based guidelines:

Several factors influence stability beyond temperature, including:

• Buffer composition - affects protein folding and aggregation propensity

• Presence of stabilizing agents - glycerol (recommended at 5-50%) significantly enhances stability

• Protein concentration - proper concentration ranges (0.1-1.0 mg/mL) help prevent aggregation

• Container material - use low-protein binding materials for storage

• Freeze-thaw cycles - each cycle progressively reduces protein integrity and activity

For long-term storage, the protein should be aliquoted into single-use volumes to minimize freeze-thaw cycles, and 50% glycerol should be added as a cryoprotectant before freezing at -80°C for maximal stability .

How do different expression systems (yeast vs. mammalian) affect the structural properties and experimental applications of Recombinant Sorex hosonoi Cytochrome b?

The choice between yeast-derived (product code CSB-YP015075FJA1) and mammalian cell-derived (product code CSB-MP015075FJA1) Recombinant Sorex hosonoi Cytochrome b can significantly impact experimental outcomes through several key differences:

• Post-translational Modifications (PTMs): Mammalian expression systems typically provide more native-like PTMs compared to yeast systems, which may produce hypermannosylation and different glycosylation patterns. This is particularly relevant for cytochrome b, which may require specific modifications for proper integration into complex III .

• Folding and Structural Authenticity: Mammalian cell-expressed proteins often demonstrate more authentic folding patterns that closely resemble the native protein configuration, potentially leading to higher biological activity in functional assays .

• Experimental Applications:

  • For structural studies and protein-protein interaction research, mammalian-expressed protein may provide more physiologically relevant results

  • For antibody production and immunological studies, both systems can be effective, but researchers should consider the importance of conformational epitopes

  • For enzymatic activity assays, the mammalian system may better recapitulate native functionality, especially when studying electron transport chain activities

• Compatibility with Downstream Applications: Despite differences in expression systems, both protein forms have comparable purity (>85% by SDS-PAGE) and can be reconstituted following similar protocols, suggesting utility across a range of standard biochemical applications .

When designing experiments, researchers should consider these expression system differences, particularly when results will be extrapolated to in vivo mammalian systems or when studying protein-protein interactions within the electron transport chain complex.

How can Recombinant Sorex hosonoi Cytochrome b be used to study electron transport chain function in comparative mammalian systems?

Recombinant Sorex hosonoi Cytochrome b provides a valuable experimental tool for comparative studies of electron transport chain (ETC) function across mammalian systems through several methodological approaches:

• Complex III Activity Assays: The protein can be incorporated into reconstituted systems to measure ubiquinol-cytochrome c reductase activity, allowing researchers to compare the catalytic efficiency of Complex III containing Sorex hosonoi cytochrome b versus other mammalian versions .

• Inhibitor Binding Studies: Research on trypanosomatid parasites has demonstrated that cytochrome b is a target for various inhibitory compounds that disrupt ETC function . The Sorex hosonoi protein can be used to assess binding affinities and inhibitory potencies of these compounds across species, potentially revealing evolutionary adaptations in inhibitor sensitivity.

• Site-Directed Mutagenesis Experiments: Specific residues in the Qi binding site of cytochrome b have been identified as critical for inhibitor interactions and drug resistance in parasites . Researchers can introduce equivalent mutations in the Sorex hosonoi protein to determine if these residues are functionally conserved across mammalian lineages.

• Species-Specific Adaptations: By comparing the functional properties of cytochrome b from Sorex hosonoi (an insectivorous mammal with high metabolic rate) with those of other mammals, researchers can investigate adaptations in ETC function related to metabolic demands, environmental pressures, or evolutionary history.

• Structural Biology Approaches: The recombinant protein can serve as a starting point for structural studies using X-ray crystallography or cryo-electron microscopy, allowing direct comparison of structural features with cytochrome b from other species.

What are the key considerations when using Recombinant Sorex hosonoi Cytochrome b as a molecular marker in phylogenetic studies?

When utilizing Recombinant Sorex hosonoi Cytochrome b or its gene sequence as a molecular marker for phylogenetic studies, researchers should consider several methodological aspects:

How can point mutations in Recombinant Sorex hosonoi Cytochrome b be engineered to study drug-binding interactions in the Qi site?

Engineering point mutations in Recombinant Sorex hosonoi Cytochrome b presents a sophisticated approach to studying drug-binding interactions at the Qi site, building on methodologies demonstrated in trypanosomatid research . This approach involves several key steps:

• Identification of Critical Residues: Research on trypanosomatid parasites has identified specific residues within the Qi site of cytochrome b that, when mutated, confer resistance to inhibitory compounds . Homologous residues in Sorex hosonoi cytochrome b can be identified through sequence alignment and structural modeling.

• Site-Directed Mutagenesis Protocol:

  • Design primers to introduce specific point mutations at the targeted Qi site residues

  • Perform PCR-based site-directed mutagenesis on the expression vector containing the Sorex hosonoi cytochrome b gene

  • Verify successful mutagenesis through sequencing

  • Express mutant proteins in either yeast or mammalian expression systems following established protocols

• Functional Characterization:

  • Assess the enzymatic activity of wild-type and mutant proteins in reconstituted complex III systems

  • Compare IC50 values of various inhibitors against wild-type and mutant proteins

  • Measure binding affinities using techniques such as isothermal titration calorimetry or surface plasmon resonance

• Structural Analysis: If possible, determine structures of wild-type and mutant proteins in complex with inhibitors to directly visualize changes in binding interactions.

Experimental evidence from trypanosomatid parasites demonstrated that cytochrome b mutations can lead to dramatic differences in inhibitor sensitivity, ranging from hypersensitivity to complete resistance depending on the specific mutation and inhibitor combination . This approach can provide valuable insights into structure-function relationships of the Qi site and inform drug development targeting cytochrome b.

What methodologies can be employed to study the role of Recombinant Sorex hosonoi Cytochrome b in adaptive evolution related to metabolic demands in shrews?

Shrews, including Sorex hosonoi, are characterized by exceptionally high metabolic rates and energy demands. Investigating adaptive evolution of cytochrome b in these species requires sophisticated methodological approaches:

• Comparative Sequence Analysis with Selection Detection:

  • Collect cytochrome b sequences from multiple shrew species with varying metabolic rates

  • Apply selection detection algorithms (PAML, HyPhy suite) to identify sites under positive selection

  • Compare selection patterns between high-metabolism shrews and related mammals with lower metabolic rates

  • Test specific hypotheses about adaptive evolution in residues associated with electron transfer efficiency

• Functional Biochemistry of Wild-type vs. Mutant Proteins:

  • Express recombinant cytochrome b proteins from multiple species or with site-directed mutations at potentially adaptive sites

  • Measure electron transfer rates and efficiency under varying temperature conditions

  • Assess proton pumping efficiency as it relates to metabolic adaptation

  • Determine oxygen consumption rates in reconstituted systems

• Integration with Physiological Data:

  • Correlate molecular variations in cytochrome b with species-specific metabolic rates

  • Examine associations between specific amino acid substitutions and adaptations to thermal environments

  • Consider the co-evolution of cytochrome b with other components of the electron transport chain

• Ancestral Sequence Reconstruction and Resurrection:

  • Infer ancestral cytochrome b sequences at key nodes in shrew phylogeny

  • Express these reconstructed ancestral proteins

  • Compare biochemical properties of ancestral and extant proteins to trace the evolution of metabolic adaptations

Research on Sorex araneus has already established methods for analyzing cytochrome b variation in shrews, demonstrating the utility of this gene for understanding population history and adaptation . These approaches can be extended to investigate the specific role of cytochrome b in the remarkable metabolic adaptations of shrews, potentially revealing molecular mechanisms underlying their extreme energy metabolism.

What are the common challenges in experiments using Recombinant Sorex hosonoi Cytochrome b and how can they be addressed?

Experiments utilizing Recombinant Sorex hosonoi Cytochrome b may encounter several technical challenges that researchers should anticipate and address through careful experimental design:

• Protein Stability Issues:

  • Challenge: Decreased activity due to protein degradation during storage or experimentation

  • Solution: Adhere strictly to storage recommendations including the addition of 5-50% glycerol, maintaining appropriate temperature conditions, and avoiding repeated freeze-thaw cycles

  • Validation: Include positive controls with fresh protein preparations to establish baseline activity levels

• Integration into Functional Complexes:

  • Challenge: Difficulty incorporating the recombinant protein into complete Complex III for functional studies

  • Solution: Consider using partial reconstitution approaches or membrane-mimetic systems (nanodiscs, liposomes) to facilitate proper protein integration

  • Validation: Verify complex formation using techniques such as blue native PAGE or analytical ultracentrifugation

• Solubility Limitations:

  • Challenge: Poor solubility due to the hydrophobic nature of cytochrome b as an integral membrane protein

  • Solution: Optimize reconstitution conditions with appropriate detergents or lipid environments; consider using the protein in its partial form which may have improved solubility characteristics

  • Validation: Monitor aggregation state using dynamic light scattering or size exclusion chromatography

• Species-Specific Antibody Recognition:

  • Challenge: Limited cross-reactivity of available antibodies with Sorex hosonoi cytochrome b

  • Solution: Develop custom antibodies using the recombinant protein as an immunogen, or target highly conserved epitopes when using commercial antibodies

  • Validation: Perform epitope mapping to identify regions of reliable antibody recognition

• Functional Differences Between Expression Systems:

  • Challenge: Variations in activity between yeast-derived and mammalian cell-derived recombinant proteins

  • Solution: When possible, compare both forms in preliminary experiments to determine which is most suitable for the specific application

  • Validation: Include appropriate controls to account for expression system-specific effects on experimental outcomes

How can researchers design experiments to reconcile contradictory data when comparing Recombinant Sorex hosonoi Cytochrome b function with cytochrome b from other species?

When confronted with contradictory results in comparative studies of cytochrome b function across species, researchers should implement a structured experimental design to resolve discrepancies:

• Systematic Comparison Framework:

  • Design experiments that simultaneously test cytochrome b from multiple species under identical conditions

  • Include both closely related species (other Sorex members) and more distant mammals to identify patterns of functional divergence

  • Employ multiple complementary assays to measure the same functional parameters, reducing method-specific biases

• Sequence-Function Correlation Analysis:

  • Identify amino acid differences between Sorex hosonoi cytochrome b and other species being compared

  • Create chimeric proteins or site-directed mutants that systematically introduce these differences to isolate the specific residues responsible for functional disparities

  • Map functional differences to specific protein domains or residues to understand structure-function relationships

• Environmental Variable Exploration:

  • Test protein function across a range of conditions (temperature, pH, ionic strength) that might reveal species-specific adaptations

  • Consider physiological differences between species (body temperature, metabolic rate) when interpreting functional differences

  • Examine whether contradictory results might reflect adaptations to different ecological niches

• Technical Variable Control:

  • Standardize protein preparation methods, ensuring equivalent purity (>85% by SDS-PAGE) across all samples

  • Use consistent reconstitution protocols with identical buffer compositions and protein concentrations

  • Ensure all proteins are in the same oligomeric state and similar conformational distributions

• Statistical Approach for Reconciliation:

  • Employ robust statistical methods including power analysis to ensure sufficient replication

  • Use meta-analysis approaches when comparing results across multiple studies

  • Consider Bayesian methods to incorporate prior knowledge and resolve apparently contradictory findings

The study of cytochrome b in Sorex araneus demonstrated how careful analysis of seemingly contradictory patterns (lack of correlation between genetic and geographic distances) could lead to novel insights about population history and colonization patterns . Similar approaches can help resolve contradictions in functional studies of cytochrome b across species.