Cellular Localization and Processing of Artificially Constructed Proteins.

大量的蛋白质和激素作为前体被合成，并通过选择性的酶裂解肽键转化为成熟的形式。枯草芽孢杆菌胞外α -淀粉酶、中性蛋白酶和碱性蛋白酶是作为前酶合成的，它们的分泌信号肽先被去除，然后分泌的前酶中的序列被加工形成成熟酶。为了分析程序的处理系统。我们构建了人工杂交基因amyE^1 - ^1pBR322^1 - ^<>amyT。在这些杂交基因中，pBR322 DNA片段编码的小肽被插入到枯草芽孢杆菌α -淀粉酶（amyE^1）的信号肽和成熟的耐热α -淀粉酶（^1amyT）之间。脂嗜热嗜热菌A631使用dll链接DNA。在构建的杂交基因中，pTUBE638质粒中的基因诱导细胞外耐热α -淀粉酶的产量提高1.7倍。枯草杆菌比亲本amyE^1 - ^1amyT基因。在大肠杆菌（pTUBE638）中，一种n端延伸的耐热α -淀粉酶积聚在细胞的周质中，在那里它保留了21个氨基酸的额外肽。相比之下，另一种n端延伸的耐热α -淀粉酶（pTUBE638）由枯草芽孢杆菌分泌，其中含有18个氨基酸的额外肽。纯化了n端延伸的耐热α -淀粉酶。杆菌(pTUBE638)。在此基础上，建立了一套体外加工系统，用于分析枯草芽孢杆菌的蛋白质加工过程。然后，我们证明了细胞外碱性蛋白酶作为主要的加工酶选择性切割ide。

A large number of proteins and hormones are synthesized as precursors and are converted into the mature forms by selective enzymatic cleavage of peptide bonds. As extracellular alpha-amylase, neutral protease and alkaline protease in Bacillus subtilis are synthesized as prepro enzymes, their signal peptides for secretion are removed at first, and then the prosequences in the secreted proenzymes are processed to form the mature enzymes. In order to analyze the processing system of the prosequences in. subtilis, we constructed artificial hybrid genes, amyE^1 - ^1pBR322^1 - ^<>amyT.In these hybrid genes, small peptides encoded in the fragments of pBR322 DNA were inserted between the signal peptide of B. subtilis alpha-amylase (amyE^1) and the mature thermostable alpha-amylase (^1amyT) of. stearothermophilus A631 using dlll linker DNA. Among the constructed hybrid genes, the gene in the plasmid pTUBE638 induced a 1.7-fold higher production of extracellular thermostable alpha-amylase in. Subtilis than by the parental amyE^1 - ^1amyT gene. In B. Coli (pTUBE638), an N-terminally extended thermostable alpha-amylase accumilates in the periplasm of the cells, where it retains the 21-amino-acid-extra-peptide. In contrast, another N-terminally extended thermostable alpha-amylase, in which an 18-amino-acid-extra-peptide, is secreted from B. Subtilis (pTUBE638). We purified the N-terminally extended thermostable alpha-amylase from. Coli (pTUBE638). Then we developed an in vitro processing system for the analysis of the protein processing in B. Subtilis. Then we demonstrated that the extracellular alkaline protease acts as the major processing enzyme for the selective cleavage of the ide.