Part:BBa_K5184021

rCtx-4

In order to eliminate spider mites, the spider venom peptide rCtx-4 is incorporated in our project to broaden the spectrum of molecular targets other venom peptides target. rCtx-4 is a neurotoxin obtained from the ctenid spider Phoneutria depilata, naturally employed to incapacitate their prey. Having the voltage-gated sodium channels playing a vital role in neuronal, muscular and cardiac functions, targets experience fast immobilization after envenomation, thus it serves as an effective pesticide. Due to the cysteine-rich nature of rCtx-4, expression strategy that exterminates inclusion bodies is required, thus a G1M5 secretion system is engineered with the SVP to achieve this. Considering future pesticidal control, G1M5-rCtx-4-his can provide future iGEM teams that dedicate in exterminating other destructive pests more choices of sustainable pesticide that could be expressed correctly in Escherichia coli.

Usage and Biology

rCtx-4 is a small cysteine-rich venom peptide derived from Phoneutria depilate, first identified in [1], it consists of 53 amino acids, including 10 Cys residues that form 5 disulfide bonds. It has a cysteine network of C1xxxC2xxxC3xC4C5xxxC6xC7xxxC8xC9xxxC10, with disulfide bridges between C1C5, C2C6, C3C10, C4C9, and C7C8 [Fig1 A&B]. In addition to the covalent disulfide bridges, there is also a pair of beta strands that run antiparallel to each other, allowing hydrogen bonds between each other and, together with the disulfide bonds, folds the protein into a highly compact conformation.

Fig1: (A) Cysteine cross-bridge structure in rCtx4 B. Secondary structure of rCtx4, by structural prediction results from AlphaFold. The cyestine residues are colored orange, displaying their side chains and the rest of the peptide in white

Such a compact conformation gives rise to an inhibitor-cysteine-knot (ICK) structure between C6 and C5, allowing the venom peptide to have inhibitory effects on its molecular target: voltage-gated sodium ion channels, and to have a highly stable structure.

Toxicity Verification

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

[1]: Vásquez-Escobar, J.; Benjumea-Gutiérrez, D.M.; Lopera, C.; Clement, H.C.; Bolaños, D.I.; Higuita-Castro, J.L.; Corzo, G.A.; Corrales-Garcia, L.L. Heterologous Expression of an Insecticidal Peptide Obtained from the Transcriptome of the Colombian Spider Phoneutria depilate. Toxins 2023, 15, 436. https://doi.org/10.3390/toxins15070436