| Protein refolding
Insoluble protein from cell lysates from a l-L culture was washed with 0.2% Triton X-100 in 50 mM Tris buffer (pH 6.8) and then dissolved in 5 mL of 6 N guanidinium hydrochloride. Two refolding protocols were attempted. The redox method (De Bernardez-Clark & Georgiou, 1991; Seeley & Young, 1991) was performed as follows. To 2.5 mL of solubilized inclusion body
protein was added 2.5 mL of 10 mM DTT in 200 mM Tris-HC1 (pH 8.0). After reduction of the solubilized protein at room temperature for 30-60 min, 1 mL of 100 mM cystine (dissolved in 0.5 N NaOH) was added to give ca. 14 mM cyst(e)ine. After incubation for 10 min to oxidize remaining DTT, this mixture was added in portions to 10 volumes of 5 mM
cysteine in 100 mM Tris-HCI, pH 8.0. The final mixture, containing 0.5-2.0 mg/mL protein, and ca. 5-6 mM cyst(e)ine was shaken in air on an orbital shaker for 24 h at room temperature. The precipitated, aggregated protein was removed by centrifugation (6,000 X g, 30 rnin), and the supernatant was dialyzed overnight against 200 mM Tris pH 8.5. The sample was concentrated and further desalted by ultrafiltration (YM10). The final concentration for ca. 50 mL was 1.5 mg/mL. This protocol was later repeated on a 4-L scale fermentation by a fourfold scaleup to obtain ca. 6 mL at 40 mg/mL. In the second method, DMSO was employed as the stabilizer and oxidant during protein refolding (Tam et al., 1991). Thus, addition of 2.5 mL of solubilized inclusion body protein above was done in 100-pL aliquots to 30 mL of 25% DMSO in 75% 100 mM MES (pH 5.6). Although a white precipitate was formed, the mixture was shaken gently for 24 h at room temperature, insoluble material was removed by centrifugation, and the supernatant
was dialyzed against 50 mM Tris, pH 6.8, and then concentrated by ultrafiltration. The final concentration for ca. 10 mL solution was 0.7 mg/mL. Refolded protein was examined for the integrity of its pheromone-binding activity by photoaffinity labeling (see below) and for preservation of antigenic regions by Western blot immunodetection. In addition, refolded protein was subjected to preparative IEF to monitor extent of correct refolding and to purify the correctly folded isoform. Gel filtration as a final step allowed removal of oligomeric forms produced during reformation of disulfide bonds. |