Structutal analysis of pro-form of subtilisin YaB

枯草杆菌蛋白酶YaB通过前体类型的前体生物合成，枯草杆菌蛋白酶BPN '也是如此。前序列在跨细胞质膜分泌的过程中被处理掉，而前形式被分泌到培养基中。然后，前体形式被自催化加工成活性酶（成熟形式），即枯草杆菌蛋白酶YaB。然而，在几种枯草杆菌蛋白酶中，没有人成功地分离出分泌的前体形式和前体形式的结构阐明。在枯草杆菌蛋白酶BPN '的情况下，有几个迹象表明前序列作为成熟形式酶的正确折叠的指导序列。1997年我们建立了枯草杆菌蛋白酶YaB前体的实验系统。当一个活性中心突变型的枯草杆菌蛋白酶YaB（Ser214Cys）编码质粒被导入到几株枯草杆菌中时，只有WB600菌株（6株胞外蛋白酶缺陷型）分泌一个分子量为52kDa的前体。在凝胶电泳过程中，推定的前体形式必须是不寻常的形状，因为检测值与估计的分子量36 kDa形成鲜明对比。不幸的是，假定的前体形式的数量很少。在1998财政年度，我们试图提高收益率，并获得更有信心的证据，即假定的亲形式是真实的亲形式。尽管进行了几次试验以改变培养条件，但推定的前体形式的产量没有提高。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.