Cellular Localization and Processing of Artificially Constructed Proteins.

人工构建蛋白质的细胞定位和加工。

• 批准号: 01470120
• 负责人: YAMANE Kunio
• 金额: $ 3.71万
• 依托单位: University of Tsukuba
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-01470120/
• 关键词: Protein processing; extracellular enzymes; fused proteins; Signal peptide; thermostable alpha-amylase; Bacillus subtilis; alkaline protease; 蛋白質のプロセシング; <Bacillus>___ー <subtilis>___ー; 枯草菌; 蛋白質の分泌; プロセシング; 人為的融合蛋白質; プレプロ構造

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.

大量的蛋白质和激素作为前体合成，并通过选择性酶促切割肽键转化为成熟形式。由于枯草芽孢杆菌的胞外α-淀粉酶、中性蛋白酶和碱性蛋白酶是作为前原酶合成的，所以首先将它们分泌的信号肽去除，然后将分泌的前酶中的前序列加工成成熟酶。为了分析中序的加工系统。在枯草芽孢杆菌中，我们构建了人工杂交基因amyE^1 - ^1pBR322^1 - ^<>amyT，在这些杂交基因中，将pBR 322 DNA片段编码的小肽插入到B的信号肽之间。枯草α-淀粉酶（amyE ^1）和成熟的耐热α-淀粉酶（^1amyT）。嗜热脂肪菌A631使用dIII接头DNA。在构建的杂合基因中，质粒pTUBE 638中的基因诱导的胞外耐热α-淀粉酶的产量比对照高1.7倍。比亲本amyE^1 - ^1amyT基因更低。在B。Coli（pTUBE 638），一种N-末端延伸的热稳定α-淀粉酶，在细胞的周质中降解，在那里它保留21个氨基酸的外肽。相反，另一种N-末端延伸的热稳定α-淀粉酶，其中18-氨基酸-外肽，从B分泌。亚克隆（pTUBE 638）。我们纯化了N-末端延伸的耐热α-淀粉酶。大肠杆菌（pTUBE 638）。在此基础上，我们建立了一套体外蛋白质加工系统，用于分析B细胞的蛋白质加工过程。低调。然后我们证明了胞外碱性蛋白酶作为选择性切割IDE的主要加工酶。

项目成果

Itoh, Y., Sumi, Nakamura, K., and Yamane, K.: "processing of an NH_2 terminally extended thernoshable αーamylase by Baciltuo subtilis protease" J. Biochem.108. 954-959 (1990)

Itoh, Y.、Sumi、Nakamura, K. 和 Yamane, K.：“枯草芽孢杆菌蛋白酶对 NH_2 末端延伸的耐热 α-淀粉酶的处理”J. Biochem.108 (1990)。

Nakamura, K., Takamatsu, H., Akiyama, Y., Ito, K., and Yamane, K.: "Complementation of the protein transport defect of an Escherichia coli secY mutant (secY24) by Bacillus subtilis secY homologue." FEBS Letters. 273 ;. 75-78 (1990)

Nakamura, K.、Takamatsu, H.、Akiyama, Y.、Ito, K. 和 Yamane, K.：“通过枯草芽孢杆菌 secY 同源物补充大肠杆菌 secY 突变体 (secY24) 的蛋白质转运缺陷。”

Itoh,Y.,Sumi,M.,Nakamura,K.,and Yamane,K.: "Processing of an NH_2ーterminally extended thermostable αーamylase by <Bacillus>___ー <subtilin>___ー alkaline protease." J.Biohem.108. 954-959 (1990)

Itoh, Y.、Sumi, M.、Nakamura, K. 和 Yamane, K.：“<芽孢杆菌>____ <枯草菌素>____ 碱性蛋白酶处理 NH_2 — 末端延伸的热稳定 α — 淀粉酶。” J.Biohem.108。954-959（1990）

Nakamura,K.,Nakamura,A.,Takamatsu,H.,Yoshikawa,H.,and Yamane,K.: "cloning and Characterization of a <Bacillus>___ー <subtilis>___ー gene homologous to E__ー.<coli>___ー <secY>___ー." J.Biochem.107. 603-607 (1990)

Nakamura, K.、Nakamura, A.、Takamatsu, H.、Yoshikawa, H. 和 Yamane, K.：“与 E__同源的 <芽孢杆菌>_____ <枯草杆菌>_____ 基因的克隆和表征.<大肠杆菌>___ー <secY>___ー。” J.Biochem.107. 603-607 (1990)

K.Nakamura,A.Nakamura,H.Takamatsu,H.Yoshikawa,K.Yamane: "Cloning and characterization of a Bacillus subtilis gene homologous to E.coli secY." J.Biochem.

K.Nakamura、A.Nakamura、H.Takamatsu、H.Yoshikawa、K.Yamane：“与大肠杆菌 secY 同源的枯草芽孢杆菌基因的克隆和表征。”