Recombinant Sambucus nigra Cytochrome c

What is cytochrome c from Sambucus nigra and why is it significant for research?

Cytochrome c is a highly conserved heme protein involved in electron transfer, cell apoptosis, and processes associated with oxidative stress. When sourced from Sambucus nigra (elderberry), it presents a unique research opportunity due to the plant's known medicinal properties. Sambucus nigra contains high levels of biologically active compounds with antioxidant, anti-inflammatory, and anticancer properties, which may influence the functional characteristics of its cytochrome c protein. Recombinant expression allows researchers to produce and study this protein in controlled laboratory conditions, enabling investigations into its structure-function relationships and potential therapeutic applications .

What are the key structural and functional domains of Sambucus nigra cytochrome c that researchers should be aware of?

Cytochrome c typically contains a single heme group covalently attached to the protein backbone via thioether bonds to cysteine residues in a CXXCH motif. This conserved structural feature is crucial for its electron transfer functionality and should be present in Sambucus nigra cytochrome c as well. The protein likely maintains the characteristic tertiary fold observed in other cytochrome c proteins, featuring α-helical segments surrounding the heme group. When expressing recombinant versions, researchers should pay particular attention to proper heme attachment during protein biogenesis, as this is essential for functional activity. The System I (CcmABCDEFGH) bacterial cytochrome c biogenesis pathway can be utilized for efficient heme incorporation during recombinant production .

What expression systems are most effective for producing recombinant Sambucus nigra cytochrome c?

Based on successful cytochrome c expression studies, E. coli represents a viable expression system for recombinant Sambucus nigra cytochrome c production. A key consideration is ensuring proper heme attachment, which can be achieved by co-expressing the bacterial System I (CcmABCDEFGH) cytochrome c biogenesis pathway. This approach facilitates the correct post-translational modification necessary for functional cytochrome c. The expression protocol typically involves transforming E. coli with plasmids containing both the Sambucus nigra cytochrome c gene and the complete Ccm operon, followed by induction under optimized conditions. This method has demonstrated yields of 10-15 mg/L for human cytochrome c and could be adapted for Sambucus nigra cytochrome c .

How can researchers optimize codon usage for efficient expression of Sambucus nigra cytochrome c in bacterial systems?

Optimizing codon usage is crucial when expressing plant proteins in bacterial systems due to different codon preferences between organisms. For Sambucus nigra cytochrome c expression in E. coli, researchers should:

• Analyze the natural codon usage in the Sambucus nigra cytochrome c gene

• Modify codons to match E. coli's preferred codons, particularly for rare amino acids

• Avoid sequential rare codons that could cause ribosomal stalling

• Consider using specialized E. coli strains that supply rare tRNAs if codon optimization isn't possible

• Ensure the optimized sequence maintains the critical CXXCH motif essential for heme attachment

This approach minimizes translational pauses and protein misfolding, thereby enhancing expression efficiency and protein quality. Codon optimization software tools can facilitate this process by providing codon adaptation index (CAI) values to guide sequence modifications .

What purification strategies yield the highest purity and activity for recombinant Sambucus nigra cytochrome c?

A multi-step purification strategy is recommended for obtaining high-purity, active recombinant Sambucus nigra cytochrome c:

• Initial extraction: Cell lysis followed by centrifugation to separate soluble cytochrome c

• Primary capture: Ion exchange chromatography (typically cation exchange) exploiting cytochrome c's positive charge at physiological pH

• Intermediate purification: Hydrophobic interaction chromatography to separate based on surface hydrophobicity

• Polishing: Size exclusion chromatography to remove aggregates and achieve final purity

• Quality control: Heme staining to confirm proper heme incorporation, essential for functional activity

Each step should be optimized with appropriate buffer conditions that maintain protein stability, particularly ensuring adequate calcium ion concentration which is critical for structural integrity. This approach typically yields protein with >95% purity suitable for subsequent biochemical and structural studies .

What spectroscopic methods are most informative for characterizing recombinant Sambucus nigra cytochrome c?

For comprehensive characterization of recombinant Sambucus nigra cytochrome c, the following spectroscopic methods are particularly informative:

• UV-Visible absorption spectroscopy: Provides confirmation of proper heme incorporation through characteristic Soret (~410 nm) and Q bands (500-550 nm). The reduced (ferrous) and oxidized (ferric) states exhibit distinctive spectral features that confirm functional integrity.

• Circular dichroism (CD): Assesses secondary structure composition and proper protein folding. The α-helical content typical of cytochrome c produces characteristic negative bands at 208 and 222 nm.

• Fluorescence spectroscopy: Monitors tryptophan fluorescence quenching by the heme group, providing information on protein conformation and heme-protein interactions.

• Nuclear magnetic resonance (NMR) spectroscopy: Offers detailed structural analysis at the atomic level, as previously demonstrated for recombinant human cytochrome c. This can reveal whether the recombinant Sambucus nigra protein exhibits the expected tertiary fold .

• Resonance Raman spectroscopy: Provides specific information about the heme environment and coordination state.

These complementary techniques provide a comprehensive assessment of structural integrity and functional capacity of the recombinant protein .

How can researchers effectively assess the redox properties of recombinant Sambucus nigra cytochrome c?

Thorough assessment of redox properties for recombinant Sambucus nigra cytochrome c requires multiple complementary approaches:

• Cyclic voltammetry: This technique directly measures the redox potential of the protein, as demonstrated with recombinant human cytochrome c (0.246 V). A three-electrode system with a working electrode modified to facilitate electron transfer with the protein provides the most reliable measurements .

• Spectroelectrochemistry: Combines spectroscopic and electrochemical measurements to monitor redox state transitions, providing insights into both thermodynamic and kinetic aspects of electron transfer.

• Electron transfer kinetics: Measures reaction rates with physiological redox partners using stopped-flow spectroscopy to assess functional competence.

• pH-dependence studies: Evaluates how the redox potential varies with pH, revealing information about proton-coupled electron transfer mechanisms.

• Temperature-dependence analysis: Provides thermodynamic parameters (ΔH°, ΔS°) associated with the redox reaction.

Together, these approaches provide a comprehensive profile of the protein's redox behavior, essential for understanding its potential functional roles and applications in research .

What techniques can determine if recombinant Sambucus nigra cytochrome c contains the proper heme incorporation and protein folding?

Proper heme incorporation and protein folding in recombinant Sambucus nigra cytochrome c can be assessed using several complementary techniques:

• Heme staining: A direct method to visualize heme-containing proteins after SDS-PAGE separation. This approach confirms the covalent attachment of heme to the protein backbone, essential for functionality .

• UV-visible absorption spectroscopy: The characteristic Soret band (~410 nm) and Q bands (500-550 nm) provide information on heme incorporation and environment. The reduced/oxidized spectra ratio can indicate proper heme coordination.

• Mass spectrometry: Precisely determines whether the protein mass includes the heme moiety and confirms the presence of expected post-translational modifications.

• Circular dichroism (CD): Assesses secondary structure elements to confirm proper folding. Cytochrome c typically exhibits strong α-helical signatures.

• Functional assays: Electron transfer activity with known redox partners provides the ultimate confirmation of proper structure and heme incorporation.

• Thermal stability analysis: Differential scanning calorimetry or thermal shift assays can reveal whether the protein exhibits the expected stability profile, indicating proper folding .

How does recombinant Sambucus nigra cytochrome c interact with bioactive compounds found in elderberry extracts?

The interaction between recombinant Sambucus nigra cytochrome c and elderberry bioactive compounds presents an intriguing research area. Elderberry extracts contain numerous bioactive molecules, particularly flavonoids such as rutin, quercetin, and kaempferol, which exhibit antioxidant properties . These compounds may interact with cytochrome c in several ways:

• Direct binding: Flavonoids may bind to specific sites on cytochrome c, potentially altering its structure or function. This could be investigated using fluorescence quenching, isothermal titration calorimetry, or NMR spectroscopy.

• Redox modulation: The antioxidant properties of elderberry compounds might influence the redox state of cytochrome c, potentially protecting it from oxidative damage or modulating its electron transfer capabilities.

• Chaperone-like effects: Some plant polyphenols exhibit chaperone-like activities that could influence cytochrome c folding or stability.

• Functional synergy: When co-present, cytochrome c and elderberry compounds might exhibit synergistic effects in biological systems that neither component shows independently.

These interactions could be systematically investigated through in vitro binding studies and functional assays to understand their biological relevance .

What roles might Sambucus nigra cytochrome c play in the plant's anti-inflammatory and antioxidant properties?

Sambucus nigra (elderberry) exhibits notable anti-inflammatory and antioxidant properties, and its cytochrome c might contribute to these effects through several potential mechanisms:

Research examining these potential mechanisms could provide insights into both the fundamental biology of Sambucus nigra and the molecular basis for its therapeutic effects .

How can researchers investigate potential pharmacological applications of recombinant Sambucus nigra cytochrome c?

Investigating the pharmacological potential of recombinant Sambucus nigra cytochrome c requires a systematic research approach:

• Oxidative stress protection assays: Evaluate the protein's ability to mitigate oxidative damage in cellular models, similar to studies with elderberry extract that demonstrated protection against reactive oxygen species in hepG2 and Caco-2 cells .

• Anti-inflammatory activity assessment: Examine effects on inflammatory signaling pathways and cytokine production in immune cell models, drawing from methodologies used to study elderberry's anti-inflammatory properties in neutrophils and macrophages .

• Cancer cell interaction studies: Investigate interactions with cancer cell lines, considering elderberry's demonstrated effects on proliferation of breast cancer (MCF7) and bladder carcinoma (T24) cells .

• Structure-function relationship studies: Identify active domains or residues unique to Sambucus nigra cytochrome c that might contribute to any observed bioactivities.

• In vitro to in vivo translation: Progress promising findings to appropriate animal models of inflammation or oxidative stress-related conditions.

• Combination studies: Examine synergistic effects when combined with other elderberry components, as the plant's therapeutic effects likely result from multiple compounds working together .

How can recombinant Sambucus nigra cytochrome c be used to study plant stress responses and adaptation mechanisms?

Recombinant Sambucus nigra cytochrome c offers a valuable tool for investigating plant stress responses and adaptation mechanisms through several experimental approaches:

• Comparative structural and functional analysis: By comparing recombinantly expressed Sambucus nigra cytochrome c with cytochrome c from other plant species, researchers can identify unique adaptations that may contribute to elderberry's renowned resilience to environmental stressors.

• Redox sensing studies: Cytochrome c's role in electron transport chain and its sensitivity to redox conditions make it an excellent model for studying how Sambucus nigra senses and responds to oxidative stress. Recombinant protein allows controlled manipulation of this system.

• Protein-protein interaction mapping: Using the recombinant protein as bait in pull-down assays or yeast two-hybrid screens can identify novel interaction partners specific to Sambucus nigra, potentially revealing unique stress response pathways.

• Transgenic expression systems: Introducing recombinant Sambucus nigra cytochrome c into model plant systems under stress conditions can reveal how this protein might confer enhanced stress tolerance.

• Evolution of stress response mechanisms: Phylogenetic analysis combined with functional characterization of recombinant cytochrome c variants can illuminate the evolutionary adaptations that contribute to Sambucus nigra's medicinal properties .

What insights might recombinantly expressed cytochrome c provide about Sambucus nigra's medicinal properties?

Recombinant expression of Sambucus nigra cytochrome c offers unique opportunities to elucidate molecular mechanisms underlying the plant's medicinal properties:

• Molecular basis of antioxidant activity: Purified recombinant cytochrome c enables detailed investigation of the protein's intrinsic antioxidant properties, potentially revealing mechanisms distinct from the plant's well-characterized phenolic compounds.

• Immune modulation pathways: Cytochrome c can trigger specific immune responses, and the Sambucus nigra variant might interact with immune cells in ways that help explain the plant's observed anti-inflammatory effects, which include inhibition of neutrophil migration and reduction of pro-inflammatory cytokines .

• Apoptosis regulation: Given cytochrome c's established role in apoptotic signaling, the Sambucus nigra protein might exhibit unique properties that contribute to the plant's observed anticancer effects on bladder carcinoma and breast cancer cell lines .

• Cultivar-specific variations: By comparing recombinant cytochrome c from different elderberry cultivars (such as Alleso, Korsor, Sampo, and Samyl), researchers might identify correlations between protein structure/function and the varying bioactive profiles documented across cultivars .

• Synergistic interactions: Studies combining recombinant cytochrome c with other elderberry components could reveal synergistic interactions that enhance bioactivity, potentially explaining why whole extracts often outperform isolated compounds .

How can researchers develop mutant libraries of recombinant Sambucus nigra cytochrome c to investigate structure-function relationships?

Developing comprehensive mutant libraries of recombinant Sambucus nigra cytochrome c requires a strategic approach:

• Rational design strategy:

  • Target evolutionarily divergent residues comparing Sambucus nigra sequence with other plant species

  • Focus on surface-exposed residues that might mediate protein-protein interactions

  • Modify residues near the heme pocket that could influence redox properties

  • Alter potential post-translational modification sites

• High-throughput mutagenesis methods:

  • Site-directed mutagenesis for specific, hypothesis-driven mutations

  • Error-prone PCR for random mutagenesis libraries

  • DNA shuffling techniques for more extensive sequence variation

  • CRISPR-based saturation mutagenesis for comprehensive coverage

• Efficient screening approaches:

  • Develop colorimetric assays based on cytochrome c's peroxidase-like activity

  • Implement redox-sensitive fluorescent reporters for rapid assessment

  • Use thermal stability assays to identify stabilizing mutations

  • Apply deep mutational scanning coupled with next-generation sequencing

• Structure-function analysis:

  • Correlate mutations with changes in redox potential measured by cyclic voltammetry

  • Assess functional changes using electron transfer kinetics assays

  • Examine structural effects through spectroscopic techniques

  • Map findings onto structural models to develop comprehensive structure-function relationships

What are common challenges in expressing recombinant Sambucus nigra cytochrome c and how can they be addressed?

Researchers face several common challenges when expressing recombinant Sambucus nigra cytochrome c, each requiring specific troubleshooting approaches:

• Insufficient heme incorporation:

  • Problem: Inadequate heme availability during expression leads to incomplete holoprotein formation

  • Solution: Supplement growth medium with δ-aminolevulinic acid (heme precursor), optimize expression conditions to match the capacity of the Ccm system, and ensure proper expression of all CcmABCDEFGH components

• Protein misfolding and aggregation:

  • Problem: Plant proteins may fold incorrectly in bacterial systems

  • Solution: Lower induction temperature (16-20°C), reduce expression rate with lower inducer concentrations, co-express molecular chaperones, or explore periplasmic expression strategies

• Low expression yields:

  • Problem: Suboptimal codon usage or toxic effects of overexpression

  • Solution: Optimize codon usage for E. coli, use tightly regulated expression systems, or explore alternative host systems like yeast

• Proteolytic degradation:

  • Problem: Susceptibility to host proteases

  • Solution: Use protease-deficient strains, include protease inhibitors during purification, or optimize extraction conditions to minimize exposure to proteolytic enzymes

• Oxidative damage during purification:

  • Problem: The heme group is sensitive to oxidative damage

  • Solution: Include reducing agents in buffers, work under nitrogen atmosphere for critical steps, and minimize exposure to light

How should researchers troubleshoot issues with recombinant Sambucus nigra cytochrome c activity and stability?

When troubleshooting activity and stability issues with recombinant Sambucus nigra cytochrome c, researchers should implement a systematic approach:

• Activity loss diagnosis:

  • Spectroscopic analysis: Check UV-visible spectrum for proper Soret band intensity and position; shifts may indicate heme degradation or miscoordination

  • Redox potential measurement: Verify using cyclic voltammetry; significant deviations from expected values suggest structural issues

  • Heme staining: Confirm covalent heme attachment using peroxidase activity-based visualization after SDS-PAGE

• Stability enhancement strategies:

  • Buffer optimization: Critical importance of calcium ions for structural stability; removal or dilution of calcium irreversibly abrogates functionality, as observed in related proteins

  • pH screening: Identify optimal pH range for maximum stability

  • Excipient screening: Test stabilizing agents like glycerol, sucrose, or specific amino acids

  • Storage condition optimization: Evaluate freeze-thaw stability and ideal storage temperature

• Functional recovery approaches:

  • Controlled denaturation-renaturation: Attempt recovery of misfolded protein

  • Heme reconstitution: For proteins with partial heme loss

  • Reducing agent treatment: For proteins with oxidized critical thiols

• Quality control implementation:

  • Establish activity benchmarks compared to other cytochrome c proteins

  • Develop thermal stability assays for batch-to-batch consistency

  • Implement long-term stability monitoring protocols

What considerations are important when scaling up production of recombinant Sambucus nigra cytochrome c for research purposes?

Scaling up production of recombinant Sambucus nigra cytochrome c for research applications requires careful attention to several key factors:

• Expression system optimization:

  • Consider transitioning from shake flasks to bioreactors for improved aeration and pH control

  • Evaluate fed-batch cultivation to achieve higher cell densities while managing metabolic burden

  • Optimize induction parameters (timing, temperature, inducer concentration) at larger scales

  • Ensure consistent co-expression of the cytochrome c biogenesis system (CcmABCDEFGH) at scale

• Process parameter considerations:

  • Dissolved oxygen tension: Critical for proper heme synthesis and incorporation

  • Temperature control: Lower temperatures (16-20°C) often improve folding but extend cultivation time

  • Media composition: Supplementation with heme precursors may be necessary

  • Induction strategy: Consider gradual inducer addition to prevent metabolic overload

• Purification scale-up challenges:

  • Increased bed heights affect chromatography resolution; consider using larger diameter columns

  • Implement tangential flow filtration for efficient concentration and buffer exchange

  • Develop robust cleaning and sanitization protocols for reusable chromatography media

  • Evaluate continuous processing options for chromatography steps

• Quality considerations:

  • Implement in-process testing to ensure consistent heme incorporation

  • Develop specifications for acceptable purity, activity, and stability

  • Establish appropriate storage conditions to maintain activity during extended storage

  • Consider protein engineering approaches to enhance stability if needed for research applications