反应详情

BMP-2 protein in 1% acetic acid or in reverse phase buffer of 0.1% TFA, 3-40% acetonitrile was dried or reduced in volume to remove acetonitrile using a speed vacuum, redissolved with four microliters of 0.01% TFA and allowed to dissolve completely for 5 to 10 minutes. The protein concentrations used were such that the final protein concentration in the folding buffer was 10 to 100 μg/ml. The folding buffer contained 100 mM buffer titrated to the appropriate pH, 5 mM EDTA, and the desired concentration of salt. Refolding of the E. coli produced BMP-2 in arginine was analyzed under reducing and non-reducing conditions using Tricine-SDS-PAGE as described above in Example 4. Substantial bands were observed at all concentrations of arginine used to refold BMP-2; however, the greatest yield of BMP-2 was obtained using 0.6 to 0.8M arginine and from 0 to 0.25M NaCl. Several types of salt were tested for ability to enhance BMP-2 refolding: NaCl, MgCl2, MgSO4, Na2SO4. Of these, NaCl and MgCl2 yielded optimal amounts of refolded BMP-2, and MgSO4 yielded intermediate amounts of refolded BMP-2. The optimal pH range for refolding BMP-2 in arginine is pH 9.5 to 10. Refolding also occurred at pH 8.5. Refolding BMP-2 in arginine was optimal at 4°, though some refolding was observed at room temperature (approximately 23°).

1. dry with materialBMP-2 protein in 1% acetic acid or in reverse phase buffer of 0.1% TFA, 3-40% acetonitrile was dried
2. customto remove acetonitrile using a speed vacuum
3. dissolutionredissolved with four microliters of 0.01% TFA
4. dissolutionto dissolve completely for 5 to 10 minutes
5. concentrationThe protein concentrations
6. concentrationprotein concentration in the folding
7. customcoli produced BMP-2 in arginine
8. customhowever, the greatest yield of BMP-2 was obtained
9. customwas optimal at 4°, though some refolding
10. customwas observed at room temperature
11. custom(approximately 23°)