TODO: update documentation to reflect changes in software used- BindCraft now does a lot of the heavy lifting, and will build a wrapper around Boltz2 instead of AlphaFold2

End to End Binder Development using Computational Tools + Synthetic Biology

This project is based on the paper De novo designed proteins neutralize lethal snake venom toxins. This specific protocol is designed around a BSL-1 lab and includes an additional experiment for designing bee antivenom using the same computational + expression system.

The aim of this experiment is to try and do it 100% automated- meaning that there can be a marketplace for producing specific products, like any other commercial marketplaces (Amazon, Coinbase, Mercor, Ethereum, Google, etc). Now, in the paper, they used AlphaFold + RFDiffusion + manual binder selection, but we should be able to automate those 3 steps with Evo2, which came out a year after their experiment.

Materials

Software: AlphaFold, RFDiffusion, BindCraft

Evo2 identifies prophage regions (regions of the genome where viruses successfully integrated themselves, whether to multiply or are still dormant). It did this by identifying specific sequences across species that had the same letters

viral markers (integrase, capsid, tail proteins)

Experimental Plan

The following experiment is based on the paper De novo designed proteins neutralize lethal snake venom toxins. The protocol is designed around a BSL-1 lab and includes an additional experiment for designing bee antivenom using the same computational + expression system.

For neutralizing the snake venom, we will follow the same process outlined in the paper to create a synthetic protein binder (SHRT) that can effectively neutralize a synthetic toxin (ScNtx for snake) and protect human cells. To do this, we will express, purify, and validate SHRT (short-chain α-neurotoxin binder) binding to ScNtx (synthetic short-chain α-neurotoxin consensus), using BSL-1 compatible tools. We will then follow the same procedure for Melittin (bee venom), as a way of expanding the current research scope.

ScNtx Antivenom

Raw SHRT binder protein sequence

>9BK7_1|Chain A|SHRT_binder|synthetic construct (32630)
MSGGPKTVVVRLSPSMNEEQAAEIGREAGKAALAAGDRLVFVGPADQSYAAMKAAMEAGLPEVTMYALDFSDAESALKAAEVAEDEGDEEVAEVAREIAEEIKAGGSGSHHWGSTHHHHHH

Codon optimized for E. Coli

AAATGCCATGCCATTAACGCGAGCCATCGCACCATTAATGATGAACGCAGCTATAATACCCATGAAACCATTTGCTGCAATAGCACCCGCTGCACCATGAGCGGCGGCCCGAAAACCGTCGTGGTGCGCCTGAGCCCGAGCATGAACGAAGAACAGGCCGCGGAAATTGGCCGCGAAGCGGGCAAAGCGGCGCTGGCCGCGGGCGATCGTCTGGTATTTGTCGGCCCGGCCGATCAGAGCTATGCCGCGATGAAAGCCGCGATGGAAGCGGGCCTGCCGGAAGTGACCATGTACGCCCTGGACTTTAGCGATGCGGAAAGCGCGCTGAAAGCGGCGGAAGTGGCGGAAGATGAAGGTGATGAAGAAGTGGCCGAAGTGGCGCGCGAAATTGCGGAAGAAATTAAAGCAGGTGGCAGCGGCAGCCATCACTGGGGTAGCACCCATCATCACCATCACCATTAA

Add Gibson overhangs

GCGCCATGGATCTGATTAAGG
AAATGCCATGCCATTAACGCGAGCCATCGCACCATTAATGATGAACGCAGCTATAATACCCATGAAACCATTTGCTGCAATAGCACCCGCTGCACCATGAGCGGCGGCCCGAAAACCGTCGTGGTGCGCCTGAGCCCGAGCATGAACGAAGAACAGGCCGCGGAAATTGGCCGCGAAGCGGGCAAAGCGGCGCTGGCCGCGGGCGATCGTCTGGTATTTGTCGGCCCGGCCGATCAGAGCTATGCCGCGATGAAAGCCGCGATGGAAGCGGGCCTGCCGGAAGTGACCATGTACGCCCTGGACTTTAGCGATGCGGAAAGCGCGCTGAAAGCGGCGGAAGTGGCGGAAGATGAAGGTGATGAAGAAGTGGCCGAAGTGGCGCGCGAAATTGCGGAAGAAATTAAAGCAGGTGGCAGCGGCAGCCATCACTGGGGTAGCACCCATCATCACCATCACCATTAA
GCTCGAGTGCGGCCGCAAGCT

Gibson-assembled into pET-28a cut with NcoI/XhoI

Protocol Summary:

Transform into BL21(DE3), 
grow in LB + Kan, 
induce at OD600 ~0.6 with 0.5 mM IPTG, 
express overnight at 20°C, 
purify via Ni-NTA, 
validate by SDS-PAGE

Melittin Antivenom

Raw Melittin-binder protein sequence

MGGSGQQAALDKVAEVLKQGATVIDAGSKLVEAAGDKVVVVGDKAGTVKTAEKGDLGDLKAVGRSGAKHHHHHH

Codon optimized for E. Coli

ATGGGCGGCAGCGGCCAGCAGGCGGCGCTGGATAAAGTGGCGGAAGTGTTAAAACAGGGCGCCACCGTTATTGATGCGGGCAGCAAACTGGTGGAAGCGGCGGGCGATAAAGTGGTTGTGGTGGGCGATAAAGCGGGCACCGTTAAAACCGCCGAAAAAGGCGATCTGGGCGATCTGAAAGCGGTGGGCCGCAGCGGCGCGAAACATCACCATCATCATCATTAA

Add Gibson overhangs

GCGCCATGGATCTGATTAAGG
ATGGGCGGCTCCGGCCAGCAGGCGGCGCTGGATAAAGTGGCGGAAGTGCTGAAACAGGGCGCGACCGTGATTGATGCGGGCTCCAAACTGGTGGAAGCGGCGGGCGATAAAGTGGTGGTGGTGGGCGATAAAGCGGGCACCGTGAAAACCGCGGAAAAAGGCGATCTGGGCGATCTGAAAGCGGTGGGCCGCTCCGGCGCGAAACATCATCATCATCATCATTAA
GCTCGAGTGCGGCCGCAAGCT

Gibson-assembled into pET-28a cut with NcoI/XhoI

Protocol summary:

BL21(DE3), 
LB + Kan, 
IPTG at OD600 ~0.6, 
20°C overnight expression, 
His-tag purification, 
validate via SDS-PAGE,
activity assay if needed

Plasmid Design

• SHRT-His in pET-28a(+): expresses SHRT binder in E. coli
• pET-28a(+) backbone has a T7 promoter and a 6xHis tag
• ScNtx in pET-28a(+): express synthetic toxin in E. coli
• Melittin-binder in pET-28a(+): test bee venom blocking binder
• Melittin: buy the synthetic peptide to test binding affinity ($85)
• sequence: P01599, GIGAVLKVLTTGLPALISWIKRKRQ

🐍 Designing the Snake Venom Binders (ScNtx Binders)

We are testing if a synthetic protein binder (SHRT) can neutralize a synthetic toxin (ScNtx or melittin) and protect human cells. To do this, we will express, purify, and validate SHRT (short-chain α-neurotoxin binder) binding to ScNtx (synthetic short-chain α-neurotoxin consensus), using BSL-1 compatible tools.

1. Synthetic Gene Design

• Use SHRT binder sequence from Fig. 2A of the paper or supplement

(Or request from corresponding author — label as: SHRT binder for ScNtx)

• Codon-optimize SHRT for E. coli expression
• Add 6xHis-tag at N-terminus for purification
• Order gene synthesized in pET-28a(+) vector

2. Expression in E. coli

• Use E. coli BL21(DE3) strain
• Transform with SHRT-pET28a plasmid
• Grow in LB + kanamycin (50 µg/mL) at 37°C
• Induce at OD600 ~0.6 with 1 mM IPTG, shift to 18°C overnight
• Harvest cells by centrifugation

3. Protein Purification

• Resuspend pellet in lysis buffer:

50 mM Tris, 300 mM NaCl, 10 mM imidazole, pH 8.0

• Lyse via sonication
• Centrifuge → collect supernatant
• Purify via Ni-NTA resin (His-tag)
• Elute with 250 mM imidazole

4. Quality Control

• Run SDS-PAGE to check size (~12–15 kDa)
• Optional: confirm folding with Circular Dichroism (CD spectrometer, Labsense)
• Buffer exchange into PBS using desalting column or spin filter

5. Binding Assay via ELISA (BSL-1)

• Purchase ScNtx peptide (synthetic consensus short-chain neurotoxin) from a peptide synthesis company (GenScript, Twist)
• need >90% purity, lyophilized form
• Coat 96-well plate with 1 µg/well ScNtx in PBS, incubate overnight at 4°C
• Block with 5% BSA in PBS for 1 hr
• Add serial dilutions of SHRT binder (in triplicates)
• Detect with anti-His-HRP + TMB substrate
• Measure absorbance at 450 nm

🐝 Designing the Bee Venom Binders (Melittin Binders)

Target:

Melittin — major bee venom peptide (26 amino acids), membrane-disrupting.

1. Synthetic Binder

• Search for melittin sequence → design binder using RFdiffusion + ProteinMPNN

Or: Evo2

• Codon-optimize → clone into pET-28a(+), same process as above

2. Purchase Synthetic Melittin

• Buy synthetic melittin from a vendor (Sigma, GenScript)
• Use same ELISA setup as ScNtx binding
• Optional: run liposome leakage assay to test functional blocking

Pipeline

1. Get target structure → Use PDB (e.g. Melittin = 2MLT) or predict if not available
2. Pick binding site → Likely the toxic active region of Melittin
3. Generate binder shape → Use a design tool to place a binder near that site
4. Design binder sequence → Convert binder backbone to sequence
5. Validate binding → Predict binder-Melittin complex and check affinity
6. Visualize → Confirm it binds in a useful way (blocks toxicity)

Summary

1. Use SHRT and ScNtx as our first system (both synthetic)
2. Express SHRT in E. coli
3. Run an ELISA to prove binding
4. For bee venom, replace ScNtx with melittin and re-run the exact protocol

Materials

ScNtx (PDB 7Z14): synthetic cytotoxin, mimics the Type IA Cytotoxin

SHRT design (PDB 9BK7): synthetic binder to neurotoxin we express through E. Coli (this is what we are testing)

α-cobratoxin (PDB 1Y15): natural neurotoxin from the Thai Cobra, binds to mouse α1 nicotinic acetylcholine receptor (nAChR) (used as a docking target in silico)

CYTX binder complex (PDB 9BK6): synthetic version of Type IA Cytotoxin (another tester, used to show broad neutralization)

Type IA Cytotoxin (PDB 5NQ4): natural toxin (just a reference structure)

Comparison