Propionic acidemia : bacterial expression system for propionyl CoA carboxylase.

丙酸血症：丙酰辅酶A羧化酶的细菌表达系统。

• 批准号: 06670762
• 负责人: OHURA Toshihiro
• 金额: $ 1.22万
• 依托单位: TOHOKU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06670762/
• 关键词: Propionic acdemia; Propiony1 CoA Carboxylase; Chaperonine

We developed a bacterial expression system for the human alpha and beta cDNAs of propionyl-CoA carboxylase (PCC). These cDNAs were co-expressed in E.coli on one plasmid vector with each cDNA having its own IPTG-inducible promoter. Only negligible amounts of active PCC were measured. Co-expression of this plasmid with a second plasmid vector overexpressing the E.coli chaperonin proteins, groES and groEL,resulted in a several hundred-fold increase in PCC Specific activity, to a level comparable to that found in crude human liver extracts. Deficiency of either alpha PCC or beta PCC results in propionic acidemia, an autosomal recessive disorder. We used this expression system to characterize one missense mutation previously described in five Japanese alleles, namely C1283T (Thr428Ile) in betaPCC.This mutant, when expressed in E.coli under the same conditions as that of wild type PCC,had null activity. This bacterial expression system can be useful for analysis of either alphaPCC or betaPCC mutations. Our findings indicated that the groES and groEL chaperonin proteins were essential for folding and assembly of the human PCC heteromeric subunits.

我们开发了一种人类丙酰辅酶a羧化酶(PCC) α和β cdna的细菌表达系统。这些cDNA在大肠杆菌的一个质粒载体上共表达，每个cDNA都有自己的iptg诱导启动子。仅测量了可忽略不计的活性PCC。该质粒与另一个表达大肠杆菌伴侣蛋白groES和groEL的质粒载体共表达，导致PCC特异性活性增加了数百倍，达到了与粗人类肝脏提取物相当的水平。α - PCC或β - PCC缺乏导致丙酸血症，一种常染色体隐性遗传病。我们使用该表达系统表征了先前在5个日本等位基因中描述的一个错义突变，即betaPCC中的C1283T （Thr428Ile）。该突变体在与野生型PCC相同的条件下在大肠杆菌中表达时，没有活性。该细菌表达系统可用于分析alphaPCC或betaPCC突变。我们的研究结果表明，groES和groEL伴侣蛋白在人类PCC异聚亚基的折叠和组装中是必不可少的。

项目成果

Ohura T et all: "A novel splicing mutation in propionic acidemia associated with a tetranucleotide direct repeat in the PCC B gene." Human Genetics. 95. 707-708 (1995)

Ohura T 等人：“丙酸血症中的一种新型剪接突变与 PCC B 基因中的四核苷酸直接重复相关。”

大浦 敏博: "プロピオン酸血症" Molecular Medicine臨時増刊、遺伝子病マニュアル(上). 32. 76-77 (1995)

Toshihiro Oura：“丙酸血症”分子医学特别版，遗传疾病手册（第 1 部分）。 32. 76-77 (1995)。

Kelson TL et all: "Chaperonin mediated folding and overexpression of recombinant human propionyl CoA carboxylase in E coli." Am J Hum Genet. 57. A244 (1995)

Kelson TL 等人：“伴侣蛋白介导重组人丙酰辅酶 A 羧化酶在大肠杆菌中的折叠和过度表达。”

Kelson T.L.et. all.: "Chaperonin mediated folding and overexpression of recombinant human propionyl CoA carboxylase in E.coli." Am J Hum Genet. 57-4. A244 (1995)

凯尔森 T.L. 等

大浦敏博: "プロピオン酸血症" Molecular Medicine臨時増刊、遺伝子病マニュアル(上). 32. 76-77 (1995)

Toshihiro Oura：“丙酸血症”分子医学特别版，遗传疾病手册（第 1 部分）。 32. 76-77 (1995)。