Recombinant Tamias cinereicollis Cytochrome c oxidase subunit 2 (MT-CO2)

Basic Research Questions

• What is Recombinant Tamias cinereicollis Cytochrome c oxidase subunit 2 (MT-CO2) and what is its biological significance?

  Recombinant Tamias cinereicollis Cytochrome c oxidase subunit 2 (MT-CO2) is a laboratory-synthesized version of a mitochondrial protein naturally found in Gray-collared chipmunks (Tamias cinereicollis) . MT-CO2 functions as a critical component of cytochrome c oxidase (Complex IV), the terminal enzyme in the mitochondrial electron transport chain that drives oxidative phosphorylation . This protein is essential for cellular respiration, catalyzing the reduction of oxygen to water using electrons from reduced cytochrome c . The recombinant form allows researchers to study specific aspects of mitochondrial function in controlled laboratory conditions without requiring tissue samples from endangered or protected species.

  The biological significance of MT-CO2 extends beyond basic respiration. It forms part of the binuclear copper A center (CuA) that accepts electrons from cytochrome c in the intermembrane space and transfers them through heme A to the active site . Research on this protein provides insights into evolutionary adaptations in mitochondrial function across different rodent species and environments.

• What expression systems are most effective for producing functional Recombinant Tamias cinereicollis MT-CO2?

  The choice of expression system significantly impacts the functionality, yield, and structural integrity of Recombinant Tamias cinereicollis MT-CO2. Common expression systems include:

  Expression System	Advantages	Disadvantages	Recommended Application
  E. coli	High yield, low cost, rapid growth	Limited post-translational modifications, inclusion body formation	Initial structural studies, antibody production
  Yeast (S. cerevisiae/P. pastoris)	Moderate eukaryotic processing, higher yield than mammalian	May not fully replicate mammalian modifications	Functional studies requiring partial activity
  Baculovirus-insect cell	Better post-translational modifications, good for membrane proteins	More complex, moderate cost	Structure-function studies requiring proper folding
  Mammalian cell lines	Most authentic modifications and folding	Lowest yield, highest cost	High-fidelity functional studies, complex assembly

  For most applications studying the electron transport function of MT-CO2, the baculovirus-insect cell system provides the best balance between authentic protein structure and reasonable yield . This system allows proper formation of the copper-binding sites crucial for electron transfer activity. When using E. coli systems, researchers should include specialized refolding protocols and copper supplementation to achieve partial functionality.

• What methods should be used to verify the purity and activity of Recombinant Tamias cinereicollis MT-CO2?

  Verification of Recombinant Tamias cinereicollis MT-CO2 quality requires multiple complementary approaches:

  Purity Assessment:

  • SDS-PAGE with Coomassie staining should show a predominant band at approximately 25.5 kDa with >90% purity

  • Western blotting using anti-MTCO2 antibodies confirms protein identity

  • Mass spectrometry verifies the exact molecular weight and can confirm amino acid sequence

  Structural Integrity:

  • Circular dichroism spectroscopy should verify expected secondary structure elements

  • UV-visible spectroscopy can detect proper incorporation of metal cofactors

  • Size-exclusion chromatography assesses aggregation state

  Functional Activity:

  • Cytochrome c oxidation assay measuring the decrease in absorbance at 550 nm

  • Oxygen consumption measurements using Clark-type electrodes

  • Electron transfer activity assessed through spectroscopic methods

  Researchers should establish baseline values for wild-type activity and compare recombinant protein performance. Typical functional recombinant preparations show at least 60-70% of the electron transfer activity observed in native mitochondrial isolates when properly reconstituted with other complex components .

• How should Recombinant Tamias cinereicollis MT-CO2 be stored to maintain stability and activity?

  Optimal storage conditions for Recombinant Tamias cinereicollis MT-CO2 vary depending on the preparation type and intended use:

  Short-term storage (1-2 weeks):

  • Store at 4°C in Tris-based buffer (pH 7.4-7.8) containing 50% glycerol as indicated in the product information

  • Include protease inhibitors to prevent degradation

  • Maintain protein concentration above 0.5 mg/ml to prevent surface adsorption losses

  Long-term storage:

  • Store at -20°C or preferably -80°C in small aliquots to avoid repeated freeze-thaw cycles

  • Add cryoprotectants such as glycerol (typically 20-50%)

  • Seal under nitrogen atmosphere to prevent oxidation of metal centers

  Stability considerations:

  • Avoid repeated freeze-thaw cycles as this can lead to up to 30% activity loss per cycle

  • Monitor the absorbance spectrum periodically to verify cofactor integrity

  • If storing in detergent-solubilized form, use concentrations above the critical micelle concentration

  Properly stored preparations typically maintain >90% activity for 6 months at -80°C, but researchers should verify activity before critical experiments, especially for older preparations.