Recombinant Canis lupus Cytochrome c oxidase subunit 2 (MT-CO2)

What is the functional role of MT-CO2 in the respiratory chain?

MT-CO2 serves as a critical subunit of cytochrome c oxidase, the terminal enzyme of the mitochondrial electron transport chain. Its primary function is to transfer electrons from cytochrome c via its binuclear copper A center to the bimetallic center of the catalytic subunit 1 . This electron transfer function is essential for the reduction of oxygen to water, which represents the final step in cellular respiration. The functional integrity of MT-CO2 is therefore critical for energy production in canid cells, making it an important target for studies of mitochondrial function and dysfunction.

How does recombinant MT-CO2 differ from the native protein?

Recombinant Canis lupus MT-CO2 is produced through an in vitro E. coli expression system , whereas the native protein is synthesized within the mitochondria. The recombinant protein typically includes a purification tag, such as the N-terminal 10xHis-tag described in the product specifications . While the amino acid sequence remains identical to the native protein, the recombinant version may lack post-translational modifications that occur in the mitochondrial environment. Additionally, the recombinant protein must be properly folded and, for functional studies, may need to be reconstituted into appropriate membrane environments to mimic its native conformation.

What expression systems are optimal for producing functional recombinant MT-CO2?

The choice should be guided by the specific research questions being addressed, with consideration for the required protein quality and functional integrity.

What purification strategies yield the highest activity for recombinant MT-CO2?

Given that commercial recombinant MT-CO2 is produced with an N-terminal 10xHis-tag , immobilized metal affinity chromatography (IMAC) represents the logical first purification step. Following IMAC, a multi-step purification protocol might include:

• Size exclusion chromatography to separate properly folded protein from aggregates

• Ion exchange chromatography for further purification if necessary

• Careful detergent selection for solubilization of this transmembrane protein

• Assessment of purity by SDS-PAGE, with commercial preparations typically achieving ≥85% purity

The critical consideration is maintaining the structural integrity of the protein throughout the purification process, which may require the inclusion of stabilizing agents in all buffers.

How can researchers verify the functional activity of purified MT-CO2?

Functional verification of purified MT-CO2 should address both structural integrity and biochemical activity. A comprehensive validation approach would include:

• Spectroscopic analysis to confirm proper folding and copper center formation

• Electron transfer assays measuring interaction with cytochrome c

• Reconstitution with other cytochrome c oxidase subunits to form functional complexes

• Oxygen consumption measurements of reconstituted complexes

• Binding assays with known interaction partners

These approaches provide complementary information about different aspects of MT-CO2 function and should be selected based on the specific research applications intended.

What storage conditions maximize the shelf life of recombinant MT-CO2?

Optimal storage of recombinant MT-CO2 requires careful attention to temperature and formulation. According to product specifications, recombinant MT-CO2 should be stored at -20°C, with extended storage recommended at -20°C or -80°C . The protein may be available in either lyophilized or liquid form, with different stability profiles:

• Liquid formulations: Shelf life of approximately 6 months at -20°C/-80°C

• Lyophilized formulations: Extended shelf life of approximately 12 months at -20°C/-80°C

Researchers should note that repeated freezing and thawing is explicitly not recommended, as this can lead to protein denaturation and loss of activity . Working aliquots should be prepared and stored at 4°C for up to one week to minimize freeze-thaw cycles .

How do buffer components affect the stability of MT-CO2 during experiments?

While specific buffer optimization data for Canis lupus MT-CO2 is not provided in the search results, general principles for transmembrane proteins suggest including:

• Physiological pH (7.2-7.4) to maintain native protein conformation

• Stabilizing agents such as glycerol (10-20%) to prevent denaturation

• Mild detergents appropriate for transmembrane proteins (e.g., n-dodecyl-β-D-maltoside)

• Protease inhibitors to prevent degradation during experimental manipulation

• Reducing agents to maintain thiol groups, though this should be carefully considered in the context of potential disulfide bonds

Researchers should empirically determine the optimal buffer composition for their specific experimental applications, particularly for functional studies where buffer components can significantly impact activity.

How does the oxygen sensitivity of Canis lupus MT-CO2 compare to cytochrome c oxidase subunits in other species?

While the search results don't provide direct information about oxygen sensitivity of Canis lupus MT-CO2 specifically, related research on cytochrome c oxidase subunit 4 provides a valuable comparative framework . Studies have shown that the COX4-2 gene appeared unresponsive to low oxygen in non-mammalian models including zebrafish, goldfish, tilapia, anoles, and turtles . This suggests possible evolutionary differences in oxygen sensing mechanisms across vertebrate lineages.

For MT-CO2 specifically, researchers should investigate whether:

• The gene contains oxygen-responsive elements (OREs) or hypoxia-responsive elements (HREs) similar to those identified in COX4-2

• Expression levels change under hypoxic conditions in different species

• Structural features that might contribute to oxygen sensitivity are conserved across species

This represents an important area for comparative analysis across canids and other mammalian orders.

What structural features of MT-CO2 are critical for interaction with other subunits of the cytochrome c oxidase complex?

Research into the structural biology of cytochrome c oxidase suggests several key features that likely determine MT-CO2 interactions with other subunits. While specific information about Canis lupus MT-CO2 interactions is limited in the search results, comparative analysis suggests attention to:

• The copper A binding domain that facilitates electron transfer

• Transmembrane regions that anchor the protein in the inner mitochondrial membrane

• Interface regions that mediate contact with subunit 1 and other components of the complex

• Conserved residues that may participate in inter-subunit communication

Analysis of these features would provide insights into both the assembly and function of the complete cytochrome c oxidase complex in Canis lupus.

How can MT-CO2 be utilized in evolutionary studies of Canidae?

The cytochrome c oxidase subunit genes have significant utility in evolutionary biology and phylogenetic analysis. Research article analysis of cytochrome c oxidase subunit I gene of Canis lupus in Türkiye demonstrates the application of these genes in population studies . For MT-CO2 specifically, researchers could:

• Conduct comparative sequence analysis across Canidae to establish evolutionary relationships

• Identify conserved and variable regions that might reflect selective pressures

• Study the co-evolution of nuclear and mitochondrial-encoded subunits of cytochrome c oxidase

• Investigate breed-specific variations within domestic dogs compared to wild canids

• Develop molecular clock analyses based on MT-CO2 sequence divergence

These approaches would contribute to understanding both the micro and macroevolutionary processes within Canidae.

What are common challenges in expressing recombinant MT-CO2 and how can they be addressed?

Expression of transmembrane proteins like MT-CO2 presents several technical challenges. Based on the information about the commercial product being expressed in E. coli , researchers should consider:

• Protein misfolding: Optimize expression temperature (typically lowering to 16-25°C) and consider fusion partners that enhance solubility

• Inclusion body formation: Develop refolding protocols or switch to expression systems more suitable for membrane proteins

• Toxicity to host cells: Use tightly regulated inducible promoters and optimize induction conditions

• Poor yield: Screen multiple expression constructs with varying tags and fusion partners

• Protein degradation: Include protease inhibitors throughout purification and handling

Each of these challenges requires systematic optimization of expression conditions, with careful validation of the final product's structural and functional properties.

How can researchers address inconsistent results in functional assays using recombinant MT-CO2?

Inconsistent results in functional assays often stem from variation in protein quality or assay conditions. To improve reproducibility, researchers should:

• Implement rigorous quality control testing of each protein preparation, including:

  • SDS-PAGE analysis to confirm purity

  • Circular dichroism to verify proper folding

  • Mass spectrometry to confirm sequence integrity

• Standardize assay conditions through:

  • Precise temperature control

  • Consistent buffer composition

  • Standardized protein concentration determination methods

  • Inclusion of both positive and negative controls in each assay

• Consider the influence of:

  • Detergent choice and concentration on protein conformation

  • Metal ion concentrations (particularly copper) on activity

  • Reconstitution methods when incorporating into membranes

Systematic attention to these factors will substantially improve reproducibility across experiments and between laboratories.

How does the sequence of Canis lupus MT-CO2 compare with that of other mammals?

While detailed cross-species comparison data is limited in the search results, the UniProt entry (P67780) for Canis lupus familiaris MT-CO2 provides a reference point for comparative analysis. Researchers investigating evolutionary patterns should consider:

• Sequence conservation in functional domains compared to variable regions

• Specific residues involved in copper binding and electron transfer

• Transmembrane domain conservation across species

• Species-specific sequence adaptations that might reflect environmental pressures

A comprehensive comparative analysis could yield insights into both the functional constraints and adaptive evolution of this essential respiratory chain component.

What experimental models are most appropriate for studying MT-CO2 dysfunction in canids?

Researchers investigating MT-CO2 dysfunction in canids should consider several model systems, each with particular advantages:

The choice of model system should be guided by the specific research questions, with consideration for both the advantages and limitations of each approach.