Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coli.
Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coli.
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DOI：
10.1186/1475-2859-9-67
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发表时间：
2010-09-13
影响因子：
6.4
通讯作者：
Ruddock LW
中科院分区：
文献类型：
--
作者：
Hatahet F;Nguyen VD;Salo KE;Ruddock LW
天然二硫键的形成是许多蛋白质的复杂且必需的翻译后修饰。这些蛋白质的大规模生产可能是困难的，并且取决于将蛋白质靶向其中天然发生二硫键形成的区室，通常是真核生物的内质网或原核生物的周质。目前认为在野生型细菌如大肠杆菌的细胞质中不可能产生大量含二硫键的蛋白质。大肠杆菌中，由于存在多种途径，为他们的减少。在这里，我们表明，Erv 1 p，巯基氧化酶和FAD依赖性催化剂的二硫键形成中发现的线粒体的膜间空间的引入，允许在异源表达的蛋白质中的天然二硫键的有效形成在细胞质中的大肠杆菌。即使不破坏参与二硫键还原的基因，例如trxB和/或戈尔，在大肠杆菌BL 21（DE 3）pLysSRARE中，活性二硫键蛋白的产量确实较高。在共表达Erv 1 p的情况下，与商业化Δ戈尔ΔtrxB菌株rosetta-gami相比，具有还原途径完整的大肠杆菌菌株的还原能力更强。我们的研究结果反驳了该领域目前的范式，即至少一种还原途径的破坏是必要的，在细胞质中的含二硫键的蛋白质的有效生产。大肠杆菌，并开辟了新的可能性，使用E。大肠杆菌作为微生物细胞工厂。
The formation of native disulfide bonds is a complex and essential post-translational modification for many proteins. The large scale production of these proteins can be difficult and depends on targeting the protein to a compartment in which disulfide bond formation naturally occurs, usually the endoplasmic reticulum of eukaryotes or the periplasm of prokaryotes. It is currently thought to be impossible to produce large amounts of disulfide bond containing protein in the cytoplasm of wild-type bacteria such as E. coli due to the presence of multiple pathways for their reduction. Here we show that the introduction of Erv1p, a sulfhydryl oxidase and FAD-dependent catalyst of disulfide bond formation found in the inter membrane space of mitochondria, allows the efficient formation of native disulfide bonds in heterologously expressed proteins in the cytoplasm of E. coli even without the disruption of genes involved in disulfide bond reduction, for example trxB and/or gor. Indeed yields of active disulfide bonded proteins were higher in BL21 (DE3) pLysSRARE, an E. coli strain with the reducing pathways intact, than in the commercial Δgor ΔtrxB strain rosetta-gami upon co-expression of Erv1p. Our results refute the current paradigm in the field that disruption of at least one of the reducing pathways is essential for the efficient production of disulfide bond containing proteins in the cytoplasm of E. coli and open up new possibilities for the use of E. coli as a microbial cell factory.
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影响因子：
56.9
作者：
Ritz, D;Lim, J;Beckwith, J
通讯作者：
Beckwith, J
DOI：
10.1073/pnas.96.24.13703
发表时间：
1999-11-23
影响因子：
11.1
作者：
Bessette, PH;Åslund, F;Georgiou, G
通讯作者：
Georgiou, G
影响因子：
16
作者：
Yamamoto, Yuii;Ritz, Dani;Poole, Leslie B.
通讯作者：
Poole, Leslie B.
影响因子：
16
作者：
Frand, AR;Kaiser, CA
通讯作者：
Kaiser, CA
影响因子：
7.7
作者：
Lange, H;Lisowsky, T;Lill, R
通讯作者：
Lill, R