E. coli cytoplasmic expression
- Construct: N-terminal His6-TEV-binder, C-terminal Avi-tag if the design is headed to BLI
- BL21(DE3), induce at OD600 ≈ 0.6-0.8 with 0.4 mM IPTG, 18 °C overnight — the low temperature trades yield for solubility, worth it for a small, disulfide-free de novo fold with no chaperone help
- Lyse in 50 mM Tris pH 8.0, 300 mM NaCl, 20 mM imidazole, 1 mM PMSF, by sonication or a homogenizer; clarify by centrifugation
Purification
- Ni-NTA IMAC: load clarified lysate, wash with lysis buffer, elute on a step or shallow gradient to 250 mM imidazole.
- TEV cleavage: dialyze the eluate into 50 mM Tris pH 8.0, 150 mM NaCl, 1 mM DTT; cleave with His-tagged TEV protease overnight at 4 °C (or 2-4 h at room temperature if the construct tolerates it).
- Reverse Ni-NTA to strip the His6 tag, uncleaved product and the His-tagged TEV itself; collect the flow-through.
- Polish by SEC (Protocol/SEC) into the final storage buffer (PBS pH 7.4) — this step doubles as the monomer/aggregate QC.
If it fails
Try periplasmic or oxidizing cytoplasmic expression (e.g. SHuffle) if the design has a disulfide, or cell-free expression for a quick solubility read before committing to a full fermentation. For therapeutic candidates, move to low-endotoxin eukaryotic expression rather than pushing E. coli further.