The renaturation efficiency of recombinant prochymosin depends on not only the renaturation condi-tions but also the solubilization (denaturation) conditions. Compared with pH 8, solubilization of prochymosin-contain-...The renaturation efficiency of recombinant prochymosin depends on not only the renaturation condi-tions but also the solubilization (denaturation) conditions. Compared with pH 8, solubilization of prochymosin-contain-ing inclusion bodies at pH 11 (8 mol/L urea) results in onefold increase of renaturation efficiency ( ~ 40% vs. ~ 20 % ). Alkaline pH facilitates the solubilization of inclusion bodies via the breakage of intermolecular disulfide bonds. Moreover, alkaline pH renders prochymosin molecules to be in a more reduced and more unfolded state which undergoes refolding readily.展开更多
The conditions (temperature, time, pH) for solubilizing inclusion bodies of prochymosin mutant, Cys45Asp/Cys50Ser, are identical with those for the wild type. Moreover, they have similar oxidative refolding behavior. ...The conditions (temperature, time, pH) for solubilizing inclusion bodies of prochymosin mutant, Cys45Asp/Cys50Ser, are identical with those for the wild type. Moreover, they have similar oxidative refolding behavior. Under the same renaturation conditions both of them can undergo correct refolding leading to the formation of activable molecules. This is quite different from the mutant with deletion of Cys250-Cys283, indicating that Cys45-Cys50 contributes less to the correct refolding of prochymosin than Cys250- Cys283. However, deletion of Cys45-Cys50 results in a remarkable decrease of the thermostability of pseudochymosin, suggesting that this disulfide bond plays an important role in stabilizing enzyme conformation. The proteolytic (P) and milk-dotting (C) activities of the mutant of pseudochymosin, Cys45Asp/Cys50Ser, are lower than those of its wild counterpart. The C/P ratio of the former is onefold higher than that of the latter.展开更多
基金Project supported by the National High Technology Programme.
文摘The renaturation efficiency of recombinant prochymosin depends on not only the renaturation condi-tions but also the solubilization (denaturation) conditions. Compared with pH 8, solubilization of prochymosin-contain-ing inclusion bodies at pH 11 (8 mol/L urea) results in onefold increase of renaturation efficiency ( ~ 40% vs. ~ 20 % ). Alkaline pH facilitates the solubilization of inclusion bodies via the breakage of intermolecular disulfide bonds. Moreover, alkaline pH renders prochymosin molecules to be in a more reduced and more unfolded state which undergoes refolding readily.
基金Project supported by the National High Technology Programme.
文摘The conditions (temperature, time, pH) for solubilizing inclusion bodies of prochymosin mutant, Cys45Asp/Cys50Ser, are identical with those for the wild type. Moreover, they have similar oxidative refolding behavior. Under the same renaturation conditions both of them can undergo correct refolding leading to the formation of activable molecules. This is quite different from the mutant with deletion of Cys250-Cys283, indicating that Cys45-Cys50 contributes less to the correct refolding of prochymosin than Cys250- Cys283. However, deletion of Cys45-Cys50 results in a remarkable decrease of the thermostability of pseudochymosin, suggesting that this disulfide bond plays an important role in stabilizing enzyme conformation. The proteolytic (P) and milk-dotting (C) activities of the mutant of pseudochymosin, Cys45Asp/Cys50Ser, are lower than those of its wild counterpart. The C/P ratio of the former is onefold higher than that of the latter.
