Omega-hexatoxin-hv1a

Introduction:

Figure 1. Omega-hexatoxin-Hv1a

This year we create a revolutionary system that integrates biological pesticide, an automatic detector, a sprinkler, and IoT. We made a database that contains most of the spider toxins and selected the target toxins by programming. Omega-hexatoxin-Hv1a is coded for the venom of a spider, Hadronyche versus.

Figure 2. Omega-hexatoxin-Hv1a structure

GreatBay-SCIE 2024

HxTx-Hv1h is a venom peptide homologous to HxTx-Hv1a and HxTx-Hv1c, sharing crucial residues of the two venom peptides. This thus allows the hybrid venom to target against both targets of the Hv1a and Hv1c: CaV and KCa respectively (voltage-gated calcium channel and calcium-dependent potassium channel) [1], which shows that Hv1h possesses enhanced efficacy although some evidence also suggests that the three venom peptides also show a high affinity towards insect nAChRs, nicotinic acetylcholine receptors [1]. This therefore allows Hv1h to be more effective against targeted pests while remaining harmless to bees (as do Hv1a and Hv1c). To further improve potency of Hv1h, a lectin, Galanthus nivalis agglutinin (GNA), is fused with Hv1h to create the recombinant Hv1h-GNA fusion protein. GNA is able to transport attached peptides across the insect gut, allowing delivery to the circulatory system [2]. Through fusion with GNA, the fusion protein Hv1h-GNA had been observed to possess both enhanced oral and contact efficacy [3]. After trying various expression system designs, we achieved successful soluble expression of Hv1h-GNA using the expression system pET28a-G1M5-His-SUMO-Hv1h-GNA-His [Fig1A], and is transformed into E. coli strain BL21(DE3). The construct is then verified by colony PCR (Fig1B&C). The culture is then harvested and has run an SDS-PAGE to verify the venom's successful expression (Fig1D).

Figure 1: A. Plasmid construct pET28a-G1M5-His-SUMO-Hv1h-GNA-His B. Colony PCR results to verify the plasmid construct C. Alignment of sequencing results of colony PCR products D. bacterial harvest of BL21(DE)3 harboring pET28a-G1M5-His-SUMO-Hv1h-GNA-His, S: supernatant, P: precipitate

The supernatant is then treated with SUMO protease, and toxicity tested against 3 groups of 20 T. urticae individuals with a spraying method, and lethality data is measured at 24, 48, and 72 hours. hv1h proved to be highly toxic against T. urticae, as demonstrated in [Fig2C&D].

Figure 2: A. T. urticae before being sprayed by culture with hv1h; B. Dead T. urticae after spraying C. Survival plot of T. urticae after being sprayed HxTx-Hv1h D. Lethality data of T. urticae over 24, 48, and 72 hours

References

[1]: Chambers, C., Cutler, P., Huang, Y., Goodchild, J. A., Blythe, J., Wang, C. K., Bigot, A., Kaas, Q., Craik, D. J., Sabbadin, D., & Earley, F. G. (2019). Insecticidal spider toxins are high affinity positive allosteric modulators of the nicotinic acetylcholine receptor. FEBS Letters, 593(12), 1336–1350. https://doi.org/10.1002/1873-3468.13435
[2] 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.
[3] Fitches, Elaine C., et al. ‘Fusion to Snowdrop Lectin Magnifies the Oral Activity of Insecticidal ω-Hexatoxin-Hv1a Peptide by Enabling Its Delivery to the Central Nervous System’. PLoS ONE, edited by Subba Reddy Palli, vol. 7, no. 6, June 2012, p. e39389. DOI.org (Crossref), https://doi.org/10.1371/journal.pone.0039389.

Mechanism of Hv1a:

According to the reference, Omega-hexatoxin-Hv1a has a structure called ICK (inhibitor cysteine knot). This kind of structure contains three disulfide bonds and beta-sheet. With this structure, Hv1a can resist the high temperature, acid base solution and the digest juice of insect gut. Hv1a can bind on insect voltage-gated Calcium channels (CaV1) in the central nervous system, making it paralyze and die eventually.^[1]

Features of Hv1a:

1. Non-toxic

Omega-hexatoxin-Hv1a is non-toxic to mammals and Hymenoptera (bees). Since the structure of the target ion channel is different, omega-hexatoxin-Hv1a does not harm mammals and bees.^[2] So it is safe to use it as a biological pesticide.

2. Biodegradable

Omega-hexatoxin-Hv1a is a polypeptide so it must degrade over time. After degradation, the toxin will become nutrition in the soil.

3. Species-specific

According to reference, Omega-hexatoxin-Hv1a has specificity to Lepidopteran (moths), Dipteran (flies) and Orthopteran (grasshoppers).

4. Eco-friendly

Compare with chemical pesticides, Omega-hexatoxin-Hv1a will not remain in soil and water so that it will not pollute the environment and won’t harm the ecosystem.

Altogether, using Omega-hexatoxin-Hv1a is totally an environmentally friendly way for solving harmful insect problems by using this ion channel inhibitor as a biological pesticide.

Target insect:

Figure 3. Hv1a target insect

Experiment

Cloning

After assembling the DNA sequences from the basic parts, we recombined toxin gene to pSB1C3 backbones and conducted a PCR experiment to check the size of each part. The DNA sequence length of these parts is around 100-150 b.p. In this PCR experiment, the toxin product's size should be near at 350-450 b.p.

Figure 4. Hv1a,
The DNA sequence length of Hv1a is around 100-150 b.p. In this PCR experiment, the product’s size should be close to 350-450 b.p.

Application of the part

1. Expressing

We chose E.coli Rosetta gami strain, which can form the disulfide bonds in the cytoplasm to express the protein. To verify the E.coli express the Hv1a with disulfide bonds, we treated the sample in two different ways. A means adding β-mercaptoethanol and sample buffer. β-mercaptoethanol can break the disulfide bonds of Hv1a and make it a linear form.

The other one adding sample buffer is the native form of Hv1a which maintains its structure. B is adding only sample buffer. The two samples are treated in boiling water for 15 mins. The SDS-PAGE shows that the native Hv1a is smaller than linear one because the disulfide bonds in Hv1a make the whole structure a globular shape.

Figure 5. Protein electrophoresis of P_T7 + RBS + Hv1a+linker+6X His-Tag (control: Without constructed plasmid)
We can see the band of Hv1a at 5-6 kDa.
A: add β-mercaptoethanol and sample buffer
B: add sample buffer

Figure 6. Protein electrophoresis of P_T7 + RBS + Hv1a+linker+Lectin+linker+6X His-Tag (control: Without constructed plasmid)
We can see the band of Hv1a-Lectin at 5-6 kDa.
A: add β-mercaptoethanol and sample buffer
B: add sample buffer

2.Purification

We sonicated the bacteria and purified the protein by 6X /pHis-Tag behind the peptide using Nickel resin column. Then we ran the SDS-PAGE to verify the purification and analyzed the concentration of Hv1a.

Figure 7.Protein electrophoresis of Hv1a-6X His-Tag purification
A is the sonication product. B is the elution product of purification.

Figure 8.Protein electrophoresis of Hv1a-linker-lectin-linker-6X His-Tag purification
A is the sonication product. B is the elution product of purification.

3.Modeling

According to reference, the energy of Ultraviolet will break the disulfide bonds and the toxicity is also decreased. To take the parameter into consideration for our automatic system, we modeled the degradation rate of the protein and modified the program in our device. Therefore, Pantide was tested under the ultraviolet light. The protein electrophoresis was shown below.

Figure 9. SDS-PAGE gel and the concentrations of UV radiolytic oxidation test to native omega-hexatoxin-Hv1a (Hv1a, 5.3 kDa). The samples are marked on the top of gel.

4. Device

We designed a device that contains detector, sprinkler, and integrated hardware with users by APP through IoT talk. We use an infrared detector to detect the number of the pest and predict what time to spray the farmland. Furthermore, other detectors like temperature, humidity, lamination, pressure of carbon dioxide and on also install in our device. At the same time, the APP would contact the users that all the information about the farmland and spray biological pesticides automatically. This device can make farmers control the farmland remotely.

Results

Pantide-expressed E. coli Rosetta gami strain and diluted it with the three concentration.We applied the sample onto the leaf disks and put five cutworms into the separate cabinets for feeding assays. The positive control in the experiment was to apply Bacillus thuringiensis, which is the most widely-used bioinsecticide. We preserved all the result of the remained leaves sealing with the glass paper and calculated the ratio of the remained area on the leaves. The collected data were analyzed by t – test. Here are the feeding assay results.

Figure 10.Above is leaves remaining area of Negative control ( DDH₂O ), Positive control ( Bacillus thuringiensis bacteria ), Hv1a+linker+6X His-Tag, Hv1a+linker+snowdrop-lectin+linker+6XHis-Tag

Figure 11.Above are leaves with of Negative control ( DDH₂O ), Positive control ( Bacillus thuringiensis bacteria ), Hv1a+linker+6X His-Tag

Reference:

1. Wang, X.H.; Connor, M.; Wilson, D.C.; Wilson, H.I.; Nicholson, G.M.; Smith, R.; Shaw, D.; Mackay, J.P.; Alewood, P.F.; Christie, M.J.; King, G.F. “Discovery and structure of a potent and highly specific blocker of insect calcium channels,” J. Biol. Chem. 2001, 276, 40306–40312
2. Monique J. Windley, Volker Herzig, Slawomir A. Dziemborowicz, Margaret C. Hardy, Glenn F. King and Graham M. Nicholson, “Spider-Venom Peptide as Bioinsecticide,” Toxins Review, 2012, 4, pp. 191-227. 3. Chambers, C., Cutler, P., Huang, Y., Goodchild, J. A., Blythe, J., Wang, C. K., Bigot, A., Kaas, Q., Craik, D. J., Sabbadin, D., & Earley, F. G. (2019). Insecticidal spider toxins are high affinity positive allosteric modulators of the nicotinic acetylcholine receptor. FEBS Letters, 593(12), 1336–1350. https://doi.org/10.1002/1873-3468.13435

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]