Sodium Chloride → HCID 178512919, compound

HRID 1609157Notes

Reaction equation

HRID 1609157 reaction equation
N(C(CO)(CO)CO)CC(O)=O.CCCCCCCCCCCCOS([O-])(=O)=O.[Na+:30].[Na+].[Cl-:32].[Mg+2].[Cl-].[Cl-].[O-]S([O-])(=O)=O.[Mg+2].[O-]S([O-])(=O)=O.[Na+].[Na+].[NH2:49][C@H:50]([C:58]([OH:60])=[O:59])[CH2:51][CH2:52][CH2:53][NH:54][C:55](=[NH:57])[NH2:56]>>[NH2:49][C@H:50]([C:58]([OH:60])=[O:59])[CH2:51][CH2:52][CH2:53][NH:54][C:55](=[NH:56])[NH2:57].[Na+:30].[Cl-:32]

Participants

7

Reactant 6

HCID 5234Sodium Chloride3×HCID 24083Sulfuric acid magnesium salt (1:1)2×HCID 24436Sodium SulfateHCID 5360315Magnesium Chloride2×HCID 23667616Tricine SDSHCID 178418600HCID 178418600, compound4×

Product 1

HCID 178512919HCID 178512919, compound

Procedure

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 0.8 M arginine and from 0 to 0.25 M 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°).

Workup

  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°)

Sources & evidence

ORD record
ord-e9f5a6de5dc542cca8113fa5f9f4f3d1
Source dataset
uspto-grants-1995_03
Patent number
US05399677

Reaction notes

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