| 2.2. Isolation and purification of IL2-PI All procedures were done at 0-4°C. Centrifugations were done at 3,000 rpm for 20 rain. Twenty grams of harvested cells were homoge- nated in 100 ml of the standard buffer (20 mM Tris-HCl pH 8.0, I mM EDTA, 0.1 rnM phenylmethanesulfonyl fluoride), supplemented with I M urea and 1% Triton X-100 and disrupted by sonic oscillation (3 cycles, 3 min each). After centrifugation, the sediment was resuspended,agitated and centrifuged successively in 25 ml of the standard buffer supplemented with 4 M urea and 2% Triton X-100, and then twice in 25 ml of the standard buffer. The sediment was solubilized with 12 ml of the standard buffer supplemented with 8 M urea (pH 8.9), stirred for 30 min and centrifuged, The supernatant was diluted with the same buffer to a final concentration of about 3 mg/ml. Cysteine groups were
converted into their S-sulfonates by oxidative sulfitolysis, accomplished by the addition of sodium sulfite and sodium tetrathionate (20 eq. sulfite/10eq, tetrathionate/1 eq.cysteine). After 6 h, the reaction was stopped by dilution with water to a final urea concentration of 2 M, and by acidification at pH 4.0 with 2 N HCI. The IL2-PI S-hexasulfon-ate was collected as an abundant white precipitate, which was washed twice with 0.01% HC1 and kept at -20°C or alternatively, it was immediately purified by anion exchange chromatography on a Q-Sepharose
Fast Flow column (Pharmacia, Sweden). The column was equilibrated in the loading buffer (8 M urea, 20 mM Tris, 0.2 M NaC1, pH 8.9), the fusion protein hexasulfonate was dissolved and loaded at a protein concentration of about 3 mg/ml and the column was eluted with an increasing NaCI gradient. IL2-PI S-hexasulfonate eluted at about 0.6 M NaC1 and then precipitated by dilution of urea to a final concentration of 2 M after acidification at pH 4.0.
2.3. Folding of the fusion protein and its enzymatic conversion into insulin IL2-PI S-hexasulfonate was dissolved in the folding buffer (50 mM glycine, 1 mM EDTA, pH 10.5) and the solution was degassed. In order to exchange the sulfonate groups and to induce disulfide bond formation, B-mercaptoethanol was added (1.5 eq. per eq. of S-sulfonate) [1]. Folding was performed at protein concentrations varying from 25 pg/ml to 1 mg/ml, at 4°C for varied times from 2 h to 24 h. To stop the reaction, the pH was adjusted to 4.0. Folding products were isolated by reversed phase HPLC, lyophilized, dissolved in 200 mM Tris, pH
9.0, to a protein concentration of 1 mg/ml and digested with trypsin (E/S: 1:600, w/w) or trypsin plus carboxypeptidase B (E/S: 1:600) at 37°C for 5 h. In order to confirm the structure of the main product, the peak corresponding to human insulin as well as a human insulin standard were cleaved with GIu-C endoproteinase at 25°C for 5 h (E/S: 1:20, w/w; in 0.2 M Tris, pH 8.0) and the digestion products were isolated by reversed phase HPLC and analyzed by fast atom bombardment mass spectrometry (FAB-MS).
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