Recombinant Helix aspersa Cytochrome c
Description
Bacterial Expression Systems
Recombinant Helix aspersa cytochrome c is synthesized using Escherichia coli expression systems coupled with cytochrome c biogenesis pathways:
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System I (CcmABCDEFGH): Enables covalent heme attachment to the CXXCH motif of apocytochrome c. This pathway ensures proper folding and heme ligation, achieving >95% purity post-purification .
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HCCS-Tethered Assays: Human HCCS has been used in vitro to attach heme to synthetic Helix aspersa apocyt c peptides (e.g., 16mer and 20mer), confirming stereochemical heme orientation via circular dichroism (CD) spectra .
Key Steps in Biogenesis
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Gene Cloning: The cyt c gene is codon-optimized for E. coli and cloned into expression vectors.
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Cotransformation: Coexpressed with System I biogenesis genes (CcmABCDEFGH) to ensure heme attachment .
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Purification: Affinity chromatography and HPLC yield holocytochrome c with <1 EU/µg endotoxin levels .
Electron Transport
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Redox Potential: +253 mV (identical to in vivo cyt c), confirmed by redox titration .
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Electron Transfer: Mediates electron shuttling between cytochrome c1 (complex III) and cytochrome oxidase (complex IV) .
Apoptotic Role
Like human cyt c, recombinant Helix aspersa cyt c triggers caspase-9 activation upon release into the cytosol, though species-specific interactions with Apaf-1 remain uncharacterized .
Evolutionary Studies
The divergent residue at position 87 and unacetylated N-terminus provide insights into mollusc-vertebrate evolutionary divergence .
Biogenesis Pathway Analysis
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Peptide Recognition: HCCS requires α-helix 1 adjacent to the CXXCH motif for heme attachment. Synthetic 16mer/20mer Helix aspersa peptides inhibit human cyt c maturation when preincubated with HCCS, highlighting conserved recognition mechanisms .
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Structural Modeling: AlphaFold3 predicts poor interaction between bacterial cyt c peptides and HCCS due to outward-facing residues (e.g., Gly in place of Phe11), validating experimental observations .
Comparative Efficiency of Biogenesis Systems
| Parameter | E. coli System I | HCCS In Vitro |
|---|---|---|
| Heme Attachment | Covalent, stereospecific | Covalent, requires α-helix 1 |
| Yield | High (mg/L scale) | Moderate (~62% release) |
| Purity | >95% (HPLC) | Requires purification |
