Goal

Cold milk is widely known to be good at cutting spice due to the binding properties of the casein protein to capsaicin. Our goal is to design a modified bovine casein protein with enhanced capsaicin-binding affinity using computational protein design tools. Express in E. coli, purify, and compare binding behavior to wild-type casein tusing biophysical and functional assays.

Design & Modeling Tools

We’ll use the full modern stack of structure- and ligand-aware design tools to maximize binding affinity:

• AlphaFold3 to fold casein and capsaicin
• Input casein (protein) and capsaicin (ligand) structures to LigandMPNN, which will try to generate sequences for the protein
• use Rosetta to fold
• Filter by:
  • RMSD of LigandMPNN structure from original casein fold to ensure faithfulness
    • LigandMPNN expects a completed, docked protein-ligand complex.
    • Autodock Vina expects PDBQT, it includes conversion files
  • Rosetta (tmol implementation) to compute protein stability and filter
  • Log-likelihood from ESM
  • Boltz-1 (basically AF3) model, input sequences
  • AutoDock Vina, input shapes
  • Compute binding poses and binding affinity; RMSD new binding pose from original binding pose
  • what is “FabFlex”

Try to order as many fragments / clonal genes as possible within budget. Casein fragment is 600bp and we expect our novel fragments to be roughly the same length.

Expression & Purification Strategy

• Vector: pET-28a(+) map, buy here on NovoPro
  • T7 promoter for strong inducible expression (a promoter that we turn on).
  • N-terminal 6×His-tag for easy purification
  • Use Ndel on 5’ for ATG and XhoI on 3’ for His-tag (look at the map)

Alternatively we can directly order a clonal gene from Twist.

• Selection Marker: Kanamycin (50 µg/mL) from pET-28a, have in lab
• Host Strain: E. coli BL21(DE3), have in lab
  • Expresses T7 RNA polymerase
  • Optimized for pET-28a (page 55) and most widely used for gene expression (page VII)
  • Compatible with kanamycin and IPTG induction

Transform with normal ice / heat shock setup, then let the cells rest and reproduce for one day. Then we activate T7 to start production.

• Induction (standard IPTG):
  • IPTG binds the lac repressor → derepresses lacUV5 → makes T7 RNA polymerase → drives transcription of gene under the T7 promoter in pET-28a(+)
  • 0.5 mM IPTG at 20°C overnight.
• Purification (standard nickel resin, buy here from ThermoFisher**)**:
  • Lyse your E. coli cells in buffer with low imidazole (e.g. 20 mM)
  • Add lysate to Ni-NTA resin in gravity column — the His-tagged casein sticks to the nickel
  • Wash with more low imidazole buffer
  • Elute your protein with high imidazole (e.g. 250 mM) — casein comes off
  • Dialysis with 10 kDa dialysis tube into a buffer for assays

Assays to Evaluate Binding

• 1. Isothermal Titration Calorimetry (ITC)
  • Gold-standard method to quantify binding constant (Kd) and thermodynamics.
  • WT casein Kd to capsaicin ~5 µM [Ma et al., 2013]; improved designs aim for sub-µM.
  • Measures heat change during capsaicin titration into protein solution.
• 2. Hydrophobic Dye Binding (ANS or Nile Red)
  • Fluorescence increases upon dye binding to hydrophobic pockets.
  • Mutant should give stronger signal than WT, indicating better capsaicin accommodation