Department of Biochemistry, Linnanmaa Campus, University of Oulu, 90570 Oulu, Finland.
Microb Cell Fact. 2010 Sep 13;9:67. doi: 10.1186/1475-2859-9-67.
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.
天然二硫键的形成是许多蛋白质的一种复杂且必要的翻译后修饰。这些蛋白质的大规模生产可能具有挑战性，并且依赖于将蛋白质靶向到天然发生二硫键形成的隔室，通常是真核生物的内质网或原核生物的周质。目前认为，由于存在多种还原途径，不可能在野生型细菌（如大肠杆菌）的细胞质中大量生产含有二硫键的蛋白质。
在这里，我们表明，引入 Erv1p，一种存在于线粒体膜间隙中的巯基氧化酶和 FAD 依赖性二硫键形成催化剂，即使不破坏参与二硫键还原的基因（例如 trxB 和/或 gor），也可以在大肠杆菌的细胞质中异源表达的蛋白质中有效地形成天然二硫键。事实上，在共表达 Erv1p 的情况下，BL21（DE3）pLysSRARE 大肠杆菌菌株（还原途径完整）中具有活性的二硫键结合蛋白的产率高于商业ΔgorΔtrxB 菌株 rosetta-gami。
我们的结果反驳了该领域目前的范式，即至少破坏一种还原途径对于在大肠杆菌的细胞质中有效生产含有二硫键的蛋白质是必不可少的，并为将大肠杆菌用作微生物细胞工厂开辟了新的可能性。
