凝乳酶原（凝乳酶）二硫键Ｃys206—Cys210的定位突变

在对凝乳酶原二硫键Ｃys206-Cys210进行定位突变过程中发现，在相应的模板序列中有自身形成自由能为－１６．１kcal/mol的茎环结构倾向，妨碍与引物结合，从而难以合成突变的ＤＮＡ，采用快退火可解决此矛盾。５个突变基因均能在大肠杆菌中高效表达，除Ｃ２０６Ａ外，约占细胞总蛋白的５０％左右，突变的复性结果表明，Ｃys206-Cys210对凝乳酶原正确折叠不是绝对必需的，但相应位置的氨基酸取代对复性效率有显著影响，在５个突变体中，Ｃ２０６Ａ／Ｃ２１０Ａ的复性率分别为Ｃ２０６Ｓ／Ｃ２１０Ｓ、Ｃ２１０Ａ、Ｃ２１０Ｓ的４．５倍、２０倍和３０倍，而C206A不能复性。Ｃ２０６Ａ／Ｃ２１０Ａ与Ｃ２０６Ｓ／Ｃ２１０Ｓ的远紫外ＣＤ光谱与野生型基本相同，其荧光发射光谱与野生型相比最大发射峰不变，而荧光强度有显著增加由于上述３个蛋白具有相同比活，说明突变分子能形成具有生物活性的空间构象，而只是某些色氨酸残基微环境受到微扰。

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.