Structutal analysis of pro-form of subtilisin YaB

与枯草杆菌毒素BPn‘一样，枯草杆菌菌素Yab是通过前PRO类型前体生物合成的。前序列在细胞质膜的分泌过程中被处理掉，并将前序列分泌到培养基中。然后，前体被自动催化处理成活性酶(成熟形式)，即枯草杆菌YAB。然而，还没有人成功地分离出分泌的前体，并在几种枯草杆菌蛋白中阐明了前体的结构。在枯草杆菌毒素BPN‘的情况下，有几个迹象表明前序列作为成熟形式酶正确折叠的引导序列。在1997财政年度，我们建立了一个获得枯草杆菌YAB原形的实验系统。当将活性中心突变的枯草杆菌YAB(Ser214Cys)编码的质粒导入几株枯草芽孢杆菌时，只有WB600菌株(6株胞外蛋白酶缺陷突变株)在SDS-凝胶电泳法中估计分泌52 kDa的原形。在凝胶电泳法的过程中，推测的前体必须是不确定的形状，因为检测到的值与估计的36 kDa的分子量形成了鲜明的对比。不幸的是，推定的支持形式的数量很少。在1998财年，我们试图提高收益率，并获得更有信心的证据，证明Putaitve Pro-Form是真正的Pro-Form。尽管进行了几次试验以改变培养条件，但推定的前体的产量并没有提高。推测的前体的结构特征仍有待阐明。

Subtilisin YaB is biologicaly synthesized via a pre-pro type precursor as is the case of subtilisin BPN'. The pre-sequence is processed off in the course of secretion across cytoplasmic membrane and the pro-form is secreted into culture media. Then the pro-form is autocatalytically processed to the active enzyme (mature form), that is subtilisin YaB.No one, however, has succeeded in the isolation of secreted pro-form and the structural elucidation of pro-form among several subtilisins. In the case of subtilisin BPN', there are several indications that the pro-sequence is acting as the guide sequence for the correct folding of mature form enzyme. In the 1997 fiscal year, we constructed an experimental system to obtain the pro-form of subtilisin YaB.When an active center mutant subtilisin YaB (Ser2l4Cys) encoding plasmid was introduced in several strains of Bacillus subtlis, only WB600 strain (6 extracellular Protease deficient mutant) secreted a putative pro-form of 52kDa as estimated in SDS-gel electrophresis. The putative pro-form must be in an unsusual shape in the course of gel electorphoresis, because the detected value is in keen contrast to the estimated molecular weight of 36 kDa. Unfortunately the amount of the putative pro-form was scarce. In the 1998 fiscal year, we tried to improve the yield and to obtain the more confident evidence that the putaitve pro-form is the real pro-form. In spite of several trials to vary the cultural condition the yield of the putative pro-form was not elevated. The stuctural characteristics of the putative pro-form has been remained to be elucidated.