[Site-directed mutagenesis at disulfide bond Cys206-Cys210 of prochymosin (chymosin)]

During the work of site-directed mutagenesis at disulfide bond Cys206-Cys210 of prochymosin, it was found that the corresponding template sequence had the potential to form a loop-stem structure with free energy of -16.1 kcal/mol, which prevent the template from pairing with primer and, in turn, the synthesis of the mutated DNA strand. Rapid annealing can overcome this difficulty. Five expression plasmids of prochymosin muants with deletion of Cys206-Cys210 (C206A, C210A, C206A/C210A, C210S and C206S/C210S) were constructed. Except for C206A they were expressed at high level in E. coli amounting to 50% of the total cellular proteins. Renaturation of the mutant prochymosin indicated that Cys206-Cys210 is dispensable for correct refolding of prochymosin. However, the amino acid residues at Cys206 and/or Cys 210 play a critical role in determining the renaturation. Among the five mutants the reactivation efficiency of C206A/C210A were about 4.5-fold, 20-fold and 30-fold higher than that of C206S/C210S, C210A and C210S respectively. C206A can not correctly refold at all. CD spectra in the far UV region indicate that C206A/C210A and C206S/C210S chymosin analogs have a secondary structure almost identical to that of the wild-type chymosin. Fluorescence spectroscopic analysis revealed that mutant chymosins have the same emission maximum at 333 nm as the wild-type chymosin but their fluorescence intensities at 333 nm are much higher than that of the wild-type chymosin. Considering that the mutants and the wild-type chymosin exhibit almost the same specific activity, it is reasonable to conclude that the mutant proteins assume a native active information with a perturbance around some tryptophan residues.