Analysis and improvement of the protein secretion pathway of Bacillus subtillis on the functional similarity between mammalian SRP 7S RNA and B.subtilis scRNA

枯草芽孢杆菌（Bacilus subtilis）和相关菌株具有产生胞外酶的强能力，所述胞外酶例如用于淀粉降解以产生葡萄糖的α-淀粉酶和用于洗涤剂的蛋白酶。为了将这种在B.subtilis中胞外酶的高生产能力应用于由B.subtilis生产人血清白蛋白、干扰素等，包括我们在内的许多实验室使用基因工程技术进行了许多实验。已分离出可产生人基因产物的枯草B转化体。但是，它们的产生量很小，并且基因产物不能作为胞外酶在培养基中积累。为了改善这种情况，已经开发了许多蛋白酶缺陷突变体。为了解决这些问题，我们分析了枯草芽孢杆菌（B.subtilis）在信号识别颗粒上的蛋白质分泌途径，发现信号识别颗粒是哺乳动物细胞的蛋白质分泌装置。B.枯草杆菌含有丰富的由271个核苷酸组成的稳定的小胞质RNA（scRNA）。其二级结构似乎与哺乳动物SRP 7S RNA（300个核苷酸）而不是大肠杆菌4.5S RNA（114个核苷酸）相当相似。我们发现scRNA与三种蛋白质形成复合物，其分子大小估计为8.2s。我们构建了scRNA和蛋白质的条件突变体，发现该复合体与蛋白质的转运和细胞生长直接相关。基于这些发现，我们希望改善枯草杆菌的蛋白质分泌。

Bacilus subtilis and relating strains have a strong ability to produce extracellular enzymes such as alpha-amylases for starch degradation to produce glucose and proteases for detergent. To apply this hyper production ability of extracellular enzymes in B.subtilis to the production of human serum albumin, interferons and others by B.subtilis, many experiments have been examined by many laboratories including us using gene engineering technology. B.subtilis transformants, by which human gene products could be produced, have been isolated. However, the produced amounts of them were small and the gene products could not be accumulated in the culture medium as the extracellular enzymes. To improve this situation, many protease-defecient mutants have been developed. But they were not sufficient to use as the host to produce those materials.To dissolve these problems, we analyzed the protein secretion pathway of B.subtilis on the signal recognition particle, which has been found as the protein secretion apparatus of mammalian cells. B.Subtilis contains stable small cytoplasmic RNA (scRNA) abundantly consisting of 271 nucleotides. Its secondary structure seems to be rather similar to mammalian SRP 7S RNA (300 nucleotides) than E.coli 4.5S RNA (114 nucleotides). We found that scRNA forms a complex with three kind of proteins, whose molecular size was estimated to be 8.2s. We then constructed conditional mutants of scRNA and proteins, we found that the complex directly relates to the protein translocation and cell growth. Based on these findings, we would like to improve the protein secretion of B.subtilis.