Recombinant Katsuwonus pelamis Cytochrome c (cyc)

What is Katsuwonus pelamis Cytochrome c and why is it significant for research?

Katsuwonus pelamis cytochrome c is a small heme-containing protein (approximately 103 amino acids) involved in the electron transport chain of skipjack tuna mitochondria. The protein's amino acid sequence (GDVAKGKKTFVQKCAQCHTVENGGKHKVGPNLWGLFGRKTGQAEGYSYTDANKSKGIVWNENTLMEYLENPKKYIPGTKMIFAGIKKKGERQDLVAYLKSATS) reveals its structural composition . This specific cytochrome c is valuable for comparative studies of electron transfer mechanisms across species, evolutionary analysis of conserved proteins, and as a model for understanding protein function in marine organisms adapted to various environmental conditions.

What expression systems are commonly used for recombinant production?

Several expression systems have proven effective for cytochrome c production:

The choice depends on research objectives, required protein modifications, and experimental design. For Katsuwonus pelamis cytochrome c, yeast expression systems have demonstrated success in producing properly folded protein with appropriate heme incorporation .

How does Katsuwonus pelamis cytochrome c compare structurally to other species?

While cytochrome c is highly conserved across species, important structural variations exist:

• Similar to other cytochromes c, the Katsuwonus pelamis variant likely adopts a globular α-helical fold with His/Met coordination of the heme iron, comparable to C. elegans cytochrome c proteins (CYC-2.1 and CYC-2.2)

• The protein contains the characteristic CXXCH motif for covalent heme attachment

• At 103 amino acids (positions 2-104), it falls within the typical size range for cytochrome c proteins

• The protein maintains the core structural elements required for electron transfer function while potentially exhibiting species-specific variations in surface residues

What are the optimal expression conditions for high-quality protein production?

For successful expression of recombinant Katsuwonus pelamis cytochrome c:

• Expression System Selection:

  • Yeast expression has proven successful, yielding protein with >90% purity

  • For E. coli expression, co-expression with the complete System I cytochrome c biogenesis pathway (CcmABCDEFGH) is essential for proper heme attachment

• Critical Parameters:

  • Signal sequence design: For E. coli expression, a chimeric construct with an E. coli-compatible N-terminal signal sequence (e.g., MetLysIleSerIleTyrAlaThrLeuAlaAlaLeuSerLeuAlaLeuProAlaGlyAla) has been effective for other cytochrome c proteins

  • Periplasmic targeting using appropriate leader sequences (like pelB) can improve maturation

  • Media supplementation with δ-aminolevulinic acid as a heme precursor

  • Lower induction temperatures (16-20°C) often improve proper folding and heme incorporation

What analytical techniques are most effective for characterizing structural integrity?

Multiple complementary techniques should be employed:

• Spectroscopic Analysis:

  • UV-visible spectroscopy to confirm characteristic cytochrome c absorption peaks (Soret band ~410 nm)

  • Circular dichroism spectroscopy to assess secondary structure and proper folding

  • Time-resolved fluorescence resonance energy transfer (TR-FRET) for conformational dynamics analysis, similar to approaches used with C. elegans cytochrome c

• Biochemical Characterization:

  • N-terminal sequencing to confirm protein identity, as performed for human cytochrome c

  • Amino acid composition analysis to verify proper translation and processing

  • Detection of post-translational modifications such as ε-N-trimethyl lysine and γ-N-monomethyl lysine, which have been observed in recombinant cytochrome c

• Functional Validation:

  • Redox potential measurements

  • Peroxidase activity assays, which have proven informative for other cytochrome c proteins

  • Electron transfer kinetics studies with physiological partners

How can conformational dynamics be investigated effectively?

Understanding cytochrome c conformational dynamics is critical for elucidating its function:

• Similar to studies with C. elegans cytochrome c, time-resolved fluorescence resonance energy transfer (TR-FRET) in dye-labeled variants can reveal coexistence of compact and extended protein species

• Different conformational populations may relate to functional properties such as peroxidase activity and interaction with binding partners

• Comparison of conformational dynamics across different species of cytochrome c can provide insights into evolutionary conservation of function

• Investigations of temperature and pH effects on conformational equilibria can reveal adaptations specific to marine species

What are effective strategies for studying protein-lipid interactions?

Cytochrome c-lipid interactions, particularly with cardiolipin (CL), are physiologically important:

• Recombinant Katsuwonus pelamis cytochrome c can be used to study interactions with CL-containing liposomes, similar to approaches with C. elegans cytochrome c

• Peroxidase activity enhancement in the presence of CL is a key functional parameter to measure

• TR-FRET techniques can characterize the conformational changes induced by CL binding

• Liposome binding assays with varying lipid compositions can identify specific lipid preferences

What purification strategies yield the highest purity protein?

Based on successful approaches with cytochrome c proteins:

• Initial Capture:

  • For His-tagged constructs, immobilized metal affinity chromatography (IMAC) provides effective initial purification

  • The characteristic red color facilitates visual tracking during purification

• Polishing Steps:

  • Ion exchange chromatography exploiting cytochrome c's typically basic pI

  • Size exclusion chromatography to remove aggregates and achieve >95% purity

  • Hydrophobic interaction chromatography as an alternative separation technique

• Quality Control:

  • SDS-PAGE analysis to confirm purity and molecular weight

  • Spectroscopic analysis of A280/A410 ratio to assess heme incorporation

  • Mass spectrometry for precise molecular weight determination and detection of modifications

How can researchers troubleshoot common expression challenges?

Common challenges and solutions include:

• Limited Heme Incorporation:

  • Ensure co-expression of complete cytochrome c maturation system (CcmABCDEFGH for E. coli)

  • Supplement growth media with heme precursors

  • Verify periplasmic targeting signal efficiency

  • Optimize induction conditions to allow sufficient time for heme incorporation

• Low Expression Yields:

  • Explore alternative signal sequences, such as those used successfully for T. thermophilus cytochrome c

  • Test different expression vectors and host strains

  • Optimize codon usage for the expression host

  • Consider dual-plasmid expression systems similar to those used for C. elegans CYC-2.1

• Protein Degradation:

  • Use protease-deficient host strains

  • Optimize cell disruption methods

  • Include appropriate protease inhibitors during purification

  • Ensure proper storage conditions to maintain protein integrity

What construct design principles maximize expression efficiency?

Key considerations for recombinant Katsuwonus pelamis cytochrome c construct design:

• Signal Sequence Selection:

  • For E. coli expression, chimeric constructs with E. coli-compatible N-terminal signal sequences have proven effective

  • The pelB leader sequence has been successfully used for periplasmic localization of cytochrome c proteins

• Tag Placement and Selection:

  • His-tag placement should minimize interference with protein folding and function

  • Consider inclusion of protease cleavage sites for tag removal if necessary for functional studies

  • The tag location (N- versus C-terminal) may affect expression and purification efficiency

• Vector Considerations:

  • For challenging cytochrome c expressions, specialized vectors like pBTR that facilitate cytochrome c expression may be beneficial

  • Co-expression of maturation factors from compatible plasmids (e.g., pEC86 containing ccmA-H) is critical for E. coli systems

How can researchers accurately assess redox properties?

Methods for comprehensive redox characterization include:

• Potentiometric Titrations:

  • Spectroelectrochemical methods to determine reduction potentials

  • Comparison with well-characterized cytochrome c proteins under identical conditions

  • Determination of temperature and pH dependence of reduction potential

• Kinetic Measurements:

  • Stopped-flow spectroscopy to measure electron transfer rates

  • Analysis of peroxidase activity, which has been informative for other cytochrome c proteins

  • Electron transfer studies with physiological partners

• Structure-Function Correlations:

  • Correlation of redox properties with structural features determined by spectroscopic methods

  • Comparison with cytochrome c proteins from other species to identify determinants of redox properties

  • Mutagenesis studies targeting residues in the heme environment to probe their contribution to redox potential

How can recombinant Katsuwonus pelamis cytochrome c be used for evolutionary studies?

Comparative analysis offers insights into protein evolution:

• Sequence analysis can identify conserved regions critical for function versus variable regions that may reflect adaptive evolution

• Comparison of biophysical properties across cytochrome c proteins from different species can reveal evolutionary constraints

• Study of temperature dependence of stability and activity may reveal adaptations specific to marine environments

• Comparative structural analysis can identify species-specific structural elements that may relate to environmental adaptation

What experimental designs are most informative for structure-function relationship studies?

Effective approaches include:

• Site-Directed Mutagenesis:

  • Systematic modification of key residues around the heme pocket

  • Alteration of surface charges to investigate protein-protein interactions

  • Introduction of probes for spectroscopic studies of protein dynamics

• Domain Swapping:

  • Creation of chimeric proteins with domains from cytochrome c of different species

  • Swapping of specific structural elements to identify determinants of functional properties

  • Analysis of sequence regions responsible for species-specific adaptations

• Post-Translational Modification Analysis:

  • Investigation of lysine methylation patterns in recombinant versus native protein

  • Assessment of how modifications affect stability, redox properties, and interactions

What emerging technologies can advance Katsuwonus pelamis cytochrome c research?

Cutting-edge approaches include:

• Advanced Structural Biology:

  • Cryo-electron microscopy for visualization of cytochrome c in different functional states

  • Hydrogen-deuterium exchange mass spectrometry for dynamics analysis

  • Integrative structural biology combining multiple techniques for comprehensive characterization

• High-Throughput Mutagenesis:

  • Deep mutational scanning to comprehensively map sequence-function relationships

  • Machine learning approaches to predict effects of mutations on function

  • Directed evolution for engineering cytochrome c with novel properties

• Cell-Free Expression Systems:

  • Development of optimized cell-free systems for rapid production and screening

  • High-throughput expression format for variant analysis

  • Direct incorporation of non-natural amino acids for specialized studies

How can comparative studies with cytochrome c from different species inform our understanding?

Multi-species analyses provide valuable perspectives:

• Comparison of Katsuwonus pelamis cytochrome c with mammalian counterparts can reveal adaptations to different physiological environments

• Analysis of temperature and pH optima across species can identify molecular determinants of environmental adaptation

• Evolutionary conservation patterns can highlight structurally and functionally critical regions

• Cross-species studies of protein-protein and protein-lipid interactions can reveal conservation of interaction mechanisms