Recombinant Acer negundo Cytochrome c

What is Acer negundo Cytochrome c and what differentiates it from other plant cytochromes?

Acer negundo (Box elder) Cytochrome c is a full-length protein (amino acids 1-112) involved in electron transport processes. While cytochrome c is generally highly conserved across species, the Acer negundo variant represents an opportunity to study this protein in the context of a North American maple species with a fully sequenced genome of 442.39 Mb . The protein's study can contribute to our understanding of plant adaptation mechanisms and electron transport systems in woody plants. The Acer negundo genome has 30,491 genes with a GC content of 34.1%, placing it at the lower range among angiosperm plants .

What expression systems are currently used for producing Recombinant Acer negundo Cytochrome c?

Recombinant Acer negundo Cytochrome c can be expressed in multiple heterologous systems including:

• Yeast

• E. coli

• Baculovirus-infected insect cells

• Mammalian cell cultures

Each system offers different advantages regarding protein folding, post-translational modifications, and yield. For bacterial expression specifically, the System I (CcmABCDEFGH) cytochrome c biogenesis pathway can be utilized in E. coli to facilitate proper heme attachment, which is crucial for producing functional holocytochrome c .

How should researchers handle and store Recombinant Acer negundo Cytochrome c for optimal stability?

For optimal stability and activity retention, researchers should follow these handling guidelines:

• Reconstitute lyophilized protein by briefly centrifuging the vial before opening

• Use sterile deionized water for reconstitution to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is recommended for maximum stability)

• Aliquot reconstituted protein to avoid repeated freeze-thaw cycles

• Store long-term at -20°C or -80°C

• Working aliquots remain stable at 4°C for up to one week

What analytical methods are appropriate for initial validation of Recombinant Acer negundo Cytochrome c?

Initial validation should include:

• SDS-PAGE to confirm purity (typically >85% for research-grade material)

• Spectrophotometric analysis at 550 nm to verify the presence of properly incorporated heme

• Heme staining following cell lysis to confirm holocytochrome c formation

• Activity assays measuring electron transfer capabilities

• Mass spectrometry to verify protein integrity and identity

How does the genomic context of Acer negundo influence cytochrome c expression compared to other Acer species?

The genomic architecture of Acer negundo provides important context for cytochrome c expression and evolution. Compared to Acer saccharum (sugar maple), A. negundo has:

• A smaller genome (442.39 Mb vs. 626.33 Mb)

• Fewer genes (30,491 vs. 40,074)

• Different chromosomal arrangements, including a large-scale translocation where two chromosomes from A. negundo are split with sections exchanged to form two different chromosomes in A. saccharum

• Lower percentage of paralogous genes

• 13 pseudo-chromosomes representing 99.74% of the genome length with high continuity (N50 of 32.30 Mb)

These genomic differences may influence regulatory mechanisms and evolutionary adaptations affecting cytochrome c expression and function, potentially reflecting different selective pressures experienced by these maple species .

What approaches should researchers use to study the electron transfer properties of Recombinant Acer negundo Cytochrome c?

Researchers investigating electron transfer properties should consider:

• Spectrophotometric assays at 550 nm to measure NADPH-cytochrome c reductase activity using standardized reaction mixtures containing approximately 40 μM cytochrome c

• Stopped-flow kinetic measurements to determine electron transfer rates

• Cyclic voltammetry to characterize redox potential

• Protein-protein interaction studies with physiological electron transfer partners

• Comparative analyses with cytochrome c from other plant species under identical experimental conditions

How can researchers differentiate between native and recombinant forms of Acer negundo Cytochrome c?

Differentiation between native and recombinant forms requires:

• Mass spectrometry to identify any expression system-specific modifications or tags

• Analysis of heme attachment efficiency and orientation

• Spectroscopic examination of the heme environment using techniques such as circular dichroism and UV-visible spectroscopy

• Functional comparison of electron transfer capabilities

• Structural analysis through techniques like X-ray crystallography or NMR to detect subtle conformational differences

The choice of expression system significantly impacts these properties, with bacterial systems like E. coli potentially lacking certain plant-specific post-translational modifications present in the native protein .

What is the recommended protocol for recombinant expression and purification of Acer negundo Cytochrome c in E. coli?

For successful expression in E. coli, researchers should:

• Co-express the cytochrome c gene with the complete System I (CcmABCDEFGH) bacterial cytochrome c biogenesis pathway components to ensure proper heme attachment

• Culture cells under conditions that balance protein expression with proper folding and heme incorporation

• Lyse cells using appropriate buffers that maintain protein stability

• Purify using a combination of techniques that may include:

  • Ion exchange chromatography

  • Size exclusion chromatography

  • Affinity chromatography if tags are incorporated

• Confirm successful holocytochrome c formation using heme staining techniques

• Verify purity by SDS-PAGE (target >85%)

• Lyophilize or store in appropriate buffer conditions with glycerol for stability

How should experiments be designed to compare functional properties of cytochrome c across different plant species including Acer negundo?

Comparative experimental designs should incorporate:

• Consistent expression systems across all species variants to minimize system-specific artifacts

• Standardized purification protocols to achieve comparable purity levels

• Identical assay conditions including temperature, pH, ionic strength, and substrate concentrations

• Spectrophotometric activity measurements at 550 nm using standardized reaction conditions

• Multiple biological and technical replicates

• Statistical analysis accounting for inter-species variation

• Complementary structural analysis to correlate functional differences with structural features

What methods are most effective for studying protein-protein interactions involving Acer negundo Cytochrome c?

For protein-protein interaction studies, researchers should consider:

• Pull-down assays using immobilized Acer negundo Cytochrome c to identify interaction partners

• Surface plasmon resonance (SPR) to determine binding kinetics and affinities

• Isothermal titration calorimetry (ITC) for thermodynamic characterization of interactions

• Cross-linking coupled with mass spectrometry to identify interaction interfaces

• Co-immunoprecipitation from plant extracts to validate physiologically relevant interactions

• Fluorescence resonance energy transfer (FRET) for real-time interaction monitoring

• Molecular docking and simulation studies to predict interaction mechanisms

How does Acer negundo Cytochrome c compare structurally and functionally to cytochrome c from model plant species?

While specific structural comparisons are not detailed in the search results, general principles suggest:

• Core structure is likely highly conserved due to the fundamental importance of cytochrome c in electron transport

• Species-specific variations may occur in surface residues affecting protein-protein interactions

• The heme-binding pocket architecture is probably preserved given its critical role in function

• Potential differences in thermal stability reflecting adaptation to different environmental conditions

• Subtle variations in redox potential that might reflect adaptation to species-specific metabolic requirements

These comparisons require experimental validation using the methodologies described in earlier sections .

What can genomic comparisons between Acer negundo and Acer saccharum reveal about cytochrome c evolution?

Genomic comparisons between these maple species provide evolutionary context:

• Acer negundo has a significantly smaller genome (442.39 Mb) compared to Acer saccharum (626.33 Mb)

• Whole genome duplication (WGD) analysis shows a single clear peak in both species at a synonymous substitution rate (Ks) consistent with the core eudicot whole genome triplication

• Acer saccharum shows evidence of a small, recent duplication peak not present in A. negundo

• Macrosynteny analysis reveals a large-scale translocation between the species, with chromosomes split and recombined

• Acer saccharum contains more gene models (40,074) than A. negundo (30,491)

• Both species have similar GC content (35.7% for A. saccharum and 34.1% for A. negundo)

These genomic differences suggest divergent evolutionary histories that may have influenced the evolution of proteins including cytochrome c .

How do different expression systems affect the properties of Recombinant Acer negundo Cytochrome c?

Expression system selection significantly impacts recombinant protein properties:

Each system provides >85% purity as verified by SDS-PAGE, but researchers should select the system most appropriate for their specific experimental requirements .

What are common challenges in recombinant expression of Acer negundo Cytochrome c and how can they be addressed?

Common challenges include:

• Incomplete heme incorporation: Ensure co-expression with appropriate cytochrome c biogenesis pathway components (System I for E. coli)

• Low yield: Optimize growth conditions, codon usage, and induction parameters

• Protein aggregation: Adjust lysis and purification buffers to maintain solubility

• Improper folding: Consider reduced induction temperature and extended expression time

• Degradation during purification: Include appropriate protease inhibitors

• Loss of activity during storage: Store with 50% glycerol at -80°C in small aliquots to prevent freeze-thaw cycles

What quality control measures are essential when working with Recombinant Acer negundo Cytochrome c?

Essential quality control measures include:

• SDS-PAGE analysis to confirm purity (target >85%)

• Spectroscopic analysis to verify proper heme incorporation

• Heme staining following cell lysis to confirm holocytochrome c formation

• Activity assays to ensure functional integrity

• Mass spectrometry to confirm protein identity and detect any modifications

• Stability testing under experimental conditions prior to use

• Regular monitoring of aliquoted samples for activity loss