Austrian Center of Industrial Biotechnology (ACIB), TU Graz, Petersgasse 14, A-8010 Graz, Austria.
Microb Cell Fact. 2010 Nov 23;9:92. doi: 10.1186/1475-2859-9-92.
Escherichia coli as a frequently utilized host organism for recombinant protein production offers different cellular locations with distinct qualities. The periplasmic space is often favored for the production of complex proteins due to enhanced disulfide bond formation, increased target product stability and simplified downstream processing. To direct proteins to the periplasmic space rather small proteinaceus tags that can be used for affinity purification would be advantageous.
We discovered that domain D of the Staphylococcus aureus protein A was sufficient for the secretion of various target proteins into the periplasmic space of E. coli. Our experiments indicated the Sec pathway as the mode of secretion, although N-terminal processing was not observed. Furthermore, the solubility of recombinant fusion proteins was improved for proteins prone to aggregation.The tag allowed a straightforward affinity purification of recombinant fusion protein via an IgG column, which was exemplified for the target protein human superoxide dismutase 1 (SOD).
In this work we present a new secretion tag that combines several advantages for the production of recombinant proteins in E. coli. Domain D of S. aureus protein A protects the protein of interest against N-terminal degradation, increases target protein solubility and enables a straight-forward purification of the recombinant protein using of IgG columns.
大肠杆菌作为重组蛋白生产的常用宿主生物，具有不同的细胞位置，具有不同的特性。由于二硫键形成增强、目标产物稳定性提高和下游处理简化，周质空间通常更适合生产复杂蛋白。为了将蛋白质导向周质空间，使用能够进行亲和纯化的较小的蛋白标签将是有利的。
我们发现金黄色葡萄球菌蛋白 A 的结构域 D 足以将各种靶蛋白分泌到大肠杆菌的周质空间中。我们的实验表明，Sec 途径是分泌的模式，尽管没有观察到 N 端加工。此外，重组融合蛋白的可溶性提高了，对于易于聚集的蛋白质。该标签允许通过 IgG 柱直接进行重组融合蛋白的亲和纯化，以目标蛋白人超氧化物歧化酶 1（SOD）为例。
在这项工作中，我们提出了一种新的分泌标签，它结合了几种在大肠杆菌中生产重组蛋白的优势。金黄色葡萄球菌蛋白 A 的结构域 D 可防止目标蛋白的 N 端降解，提高目标蛋白的可溶性，并可使用 IgG 柱直接纯化重组蛋白。
