Difference between revisions of "Part:BBa K5184074"

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G1M5 tag-rCtx-GNA

Abstract

This composite part is an expression box for the basic part rCtx-4 (BBa_K5184032), which allows the rCtx-4-GNA fusion protein, with high contact and oral toxicity, to achieve soluble expression in E. coli. This composite part includes a G1M5-SUMO (BBa_K5184022) compound tag is annexed to the N-terminus of the fusion protein, which facilitates the protein's correct folding and therefore its soluble expression. GNA, Galanthus nivalis agglutinin (BBa_K1974020) is attached to the C-terminus of the venom peptide, protecting it and conducting it in and through insect guts; achieving high oral and contact toxicity.

Usage and Biology

G1M5 is the mutated, less hydrophobic version of the secretion signal peptide of the G1 cyclomaltodextrin glucanotransferase (CGtase) of Bacillus sp., which allows the extracellular secretion of the bacterial enzyme. Conduction of proteins attached by G1M5 out of the cytosol is achieved by the Sec pathway, a very common secretion system seen in all three major domains of life: arachaea, prokaryote, and eukaryotes. Once the signal peptide, in this case G1M5 is synthesized, the protein chaperon SecB binds to the preprotein (that is attached to G1M5), and transfers the preprotein to the protein translocase SecA, of which binds to the membrane bound protein conducting channel SecYEG. Once bound to the membrane, SecA binds to a molecule of ATP, of which is hydrolyzed to conduct the protein through heterotrimer complex of SecYEG. A membrane bound SPaseI then, once enough of the preprotein had been conducted through the channel, will remove the SP and allow the preprotein to fold properly into the correct protein.[1] The in vivo SUMO tags, or small ubiquitin-like modifiers, are a diverse class of ubiquitin-related proteins that allow a versatile range of operations to be carried on a tagged protein. Amongst many of these is the selective proteolysis that removes the SUMO tag from the attached protein. Its ability to be detected and removed with high precision leads to the creation of the recombinant SUMO tag: a SUMO tag that, while different enough to the wildtype SUMO tag to not be degraded within eukaryotic hosts, can be detected and have the SUMO removed by special recombinant SUMO proteases. The recombinant SUMO therefore presents themselves as precise markers for points for proteolytic digestion that can remove attached (to SUMO tag) protein domains. SUMO tags also share high level of homology with the ubiquitin, and therefore, once synthesized, folds rapidly to hide its hydrophobic cores. It is proposed that this initial folding assists in the correct folding of the attached fusion protein by supplying a great initial "kinetic push". While this can explain SUMO tag's ability to facilitate protein folding and enhance soluble expression, there are also theories proposing the SUMO tag to work as a chaperone to assists in the attached protein's folding.[2]

rCtx-4 is a small cysteine-rich venom peptide derived from Phoneutria depilate, first identified in [3], 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.

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.

Galanthus nivalis agglutinin (GNA), a plant derived mannose-specific lectin that provides resistance to enzyme-meditated proteolytic activity in insect gastrointestinal tract is introduced and fused with the venom peptides. GNA is able to transport attached peptides across the insect gut, allowing delivery to the circulatory system. Thus, GNA allows enhanced oral and contact toxicity for the fusion proteins.[4]

Sequence and Features

Toxicity Verification

rCtx4 is synthesized using the vector pET28a-G1M5-His-SUMO-rCtx4-GNA-His[Fig2B], of which is assembled using GoldenGate cloning and transformed into E. coli strain DH5ɑ. Colony PCR and sequencing is then carried out to verify the plasmid construct, of which is extracted and transformed into BL21(DE3) strain for expression. After IPTG induction and overnight incubation, the liquid culture is harvested and, after cell lysis, have an SDS-PAGE run. The results suggest that rCtx4 had achieved soluble expression. After several unsuccessful purification attempts, the supernatant is treated directly by SUMO protease [Fig2C].

The SUMO-digested supernatant's toxicity against T. urticae females is tested using a spraying method by Professor Huang from SCAU. Results from the toxicity assay suggests rCtx4 to be highly toxic against T. urticae, achieving an astonishing fatality of 96.49% within 24 hours and complete elimination after another day [Fig2C&D].

Reference

[1]Liu, Changjun, et al. ‘A Secretory System for Extracellular Production of Spider Neurotoxin Huwentoxin-I in Escherichia Coli’. Preparative Biochemistry & Biotechnology, vol. 53, no. 8, Sept. 2023, pp. 914–22. DOI.org (Crossref), https://doi.org/10.1080/10826068.2022.2158473

[2]Jürgen Dohmen, R. ‘SUMO Protein Modification’. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, vol. 1695, no. 1–3, Nov. 2004, pp. 113–31. DOI.org (Crossref), https://doi.org/10.1016/j.bbamcr.2004.09.021

[3]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

[4]Sukiran, Nur Afiqah, et al. “Enhancing the Oral and Topical Insecticidal Efficacy of a Commercialized Spider Venom Peptide Biopesticide via Fusion to the Carrier Snowdrop Lectin ( Galanthus Nivalis Agglutinin).” Pest Management Science, vol. 79, no. 1, Jan. 2023, pp. 284–94. DOI.org (Crossref), https://doi.org/10.1002/ps.7198