Construction of a low-temperature protein expression system using a cold-adapted bacterium, Shewanella sp strain Ac10, as the host
Construction of a low-temperature protein expression system using a cold-adapted bacterium, Shewanella sp strain Ac10, as the host
复制标题
DOI：
10.1128/aem.00824-07
复制
发表时间：
2007-08-01
影响因子：
4.4
通讯作者：
Esaki, Nobuyoshi
中科院分区：
文献类型：
--
作者：
Miyake, Ryoma;Kawamoto, Jun;Esaki, Nobuyoshi
在低温下工作的重组蛋白表达系统预计可用于生产不耐热蛋白。我们利用南极冷适应细菌希瓦氏菌构建了低温表达系统。菌株Ac10作为宿主。我们评估了该细菌在 4 摄氏度下大量产生的蛋白质的启动子，以表达外源蛋白质。我们使用 27 个启动子和一个广泛宿主范围的载体 pJRD215 在希瓦氏菌中产生β-内酰胺酶。菌株Ac10。当使用推定的烷基氢过氧化物还原酶（AhpC）的启动子并且重组细胞生长至稳定期晚期时获得最大产量。 4 摄氏度下的产量为 91 毫克/升培养物，18 摄氏度下的产量为 139 毫克/升培养物。我们使用该系统从嗜冷细菌 Desulfotalea psychrophild DSM12343 中生产推定的肽酶 PepF、LAP 和 PepQ 以及推定的葡萄糖苷酶 BgIA。我们在可溶性级分中分别获得了 48、7.1、28 和 5.4 毫克/升培养物的这些蛋白质。该系统产生的PepF和PepQ的量大于大肠杆菌T7启动子系统产生的量。
A recombinant protein expression system working at low temperatures is expected to be useful for the production of thermolabile proteins. We constructed a low-temperature expression system using an Antarctic cold-adapted bacterium, Shewanella sp. strain Ac10, as the host. We evaluated the promoters for proteins abundantly produced at 4 degrees C in this bacterium to express foreign proteins. We used 27 promoters and a broad-host-range vector, pJRD215, to produce P-lactamase in Shewanella sp. strain Ac10. The maximum yield was obtained when the promoter for putative alkyl hydroperoxide reductase (AhpC) was used and the recombinant cells were grown to late stationary phase. The yield was 91 mg/liter of culture at 4 degrees C and 139 mg/liter of culture at 18 degrees C. We used this system to produce putative peptidases, PepF, LAP, and PepQ, and a putative glucosidase, BgIA, from a psychrophilic bacterium, Desulfotalea psychrophild DSM12343. We obtained 48, 7.1, 28, and 5.4 mg/liter of culture of these proteins, respectively, in a soluble fraction. The amounts of PepF and PepQ produced by this system were greater than those produced by the Escherichia coli T7 promoter system.