ShhNC24II expression and purification
This protocol is extracted from research article:
Structural basis of sterol recognition by human hedgehog receptor PTCH1
Sci Adv, Sep 18, 2019; DOI: 10.1126/sciadv.aaw6490

The synthetic DNA fragment (Genewiz) encoding the human ShhNC24II protein (residues 25 to 197, modified at the N terminus with a Cys24-Ile-Ile substitution; UniProt ID: Q15465), fused with the N-terminal 6xHis-SUMO tag, was cloned into pET28a plasmid. The plasmid was transformed into BL21(DE3) RIPL E. coli cells. The transformed cultures were grown in a shaking incubator at 37°C until OD600 of 0.8, at which point the temperature was switched to 30°C and the expression was induced with 1 mM isopropyl-β-d-1-thiogalactopyranoside (IPTG). After 3 hours, cells were collected by centrifugation, frozen, and stored at −80°C until the day of the experiment.

ShhNC24II-expressing E. coli pellets (0.5-liter culture) were thawed, resuspended in 10 ml of buffer C [50 mM tris-HCl (pH 7.5), 200 mM NaCl] containing 25 mM imidazole, disrupted by sonication, and cleared using centrifugation with a benchtop Eppendorf centrifuge for 30 min at 25,000g. The supernatant was added to 0.5 ml of NiNTA resin, incubated with rotation for 1 hour. The resin was collected on a gravity column (Bio-Rad), washed with 30 column volumes of buffer C containing 25 mM imidazole, followed by 10 column volumes of buffer C containing 50 mM imidazole. The protein was eluted with buffer C containing 250 mM imidazole. The eluted fractions were pooled, diluted eightfold to reduce the concentration of imidazole, and incubated with 250 μg of purified SUMO protease Ulp1 overnight at 4°C. The mixture was passed through 1 ml of immobilized NiNTA resin to remove uncleaved protein, excess cleaved SUMO tag, and Ulp1, and the flow-through was concentrated to 1 ml and applied to a Superdex 200 Increase 10/300 GL column. The fractions corresponding to ShhNC24II were pooled, supplemented with 10% glycerol, aliquoted into 100-μl batches, flash frozen in liquid nitrogen, and stored at −80°C. Prior to cryo-EM grid freezing, the protein was thawed, desalted into buffer B, and concentrated to a final concentration of ~76 μM using an Amicon Ultra-4 concentrator (10-kDa cutoff, Millipore).

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