摘要
在对凝乳酶原二硫键Cys2 0 6 Cys2 10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C2 0 6A外 ,约占细胞总蛋白的 5 0 %左右。突变体的复性结果表明 ,Cys2 0 6 Cys2 10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 ,C2 0 6A/C2 10A的复性率分别为C2 0 6S/C2 10S、C2 10A、C2 10S的 4 5倍、2 0倍和 30倍 ,而C2 0 6A完全不能复性。C2 0 6A/C2 10A与C2 0 6S/C2 10S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。
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.
出处
《生物工程学报》
CAS
CSCD
北大核心
2001年第1期7-10,共4页
Chinese Journal of Biotechnology
基金
国家自然科学基金资助项目!(39870 173)&&
