Department of Chemical Engineering, Feng Chia University , 100 Wenhwa Road, Taichung 40724, Taiwan.
J Agric Food Chem. 2011 Jun 22;59(12):6534-42. doi: 10.1021/jf2013748. Epub 2011 May 26.
The L-arabinose (Ara)-controlled T7 expression system was previously constructed by creation of an Escherichia coli BL21(BAD) strain. The production of recombinant proteins in this strain was stringently regulated and reached a high level upon induction with Ara. Nevertheless, this system is still associated with inherent problems of interference with glucose and of the all-or-nothing induction profile at a subsaturating level of Ara. In this study, these problems were circumvented by modifying the physiological traits of BL21(BAD) strain. This was followed by deletion of ptsG gene and the araFGH and araBAD operon. The former encodes the glucose transporter while the latter two gene operons produce proteins responsible for Ara uptake and catabolism. In addition, the expression of genomic araE (encodes the Ara transporter) was constitutively enhanced. The resulting strain was designated BAD-5. By expression of the faster degrader GFP(LAA) at a subsaturating level of Ara, 80% of BAD-5 strain was found visually bright in the presence or absence of glucose. A further analysis by flow cytometry showed a uniform distribution of GFP expression for BAD-5 strain. In marked contrast, BL21(BAD) strain exhibiting visual brightness was less than 10% of the cell population and remained dark in the presence of glucose. Moreover, a saturated level of luciferase from Renilla reniformis (Rluc) could be readily obtained in BAD-5 strain at 20 μM Ara regardless of glucose. Rluc in BL21(BAD) strain was produced in an Ara dose-dependent manner, and the protein production became arrested when glucose was present. Overall, it illustrates the usefulness of the improved system for overproduction of recombinant proteins in an efficient, homogeneous, and glucose-insensitive way.
L-阿拉伯糖（Ara）控制的 T7 表达系统以前是通过构建大肠杆菌 BL21（BAD）菌株来构建的。在该菌株中生产重组蛋白受到严格调控，并用 Ara 诱导时可达到很高水平。然而，该系统仍然存在与葡萄糖干扰和亚饱和Ara 水平下的全有或全无诱导谱相关的固有问题。在这项研究中，通过修改 BL21（BAD）菌株的生理特性来规避这些问题。随后删除了 ptsG 基因和 araFGH 和 araBAD 操纵子。前者编码葡萄糖转运蛋白，而后者两个基因操纵子产生负责 Ara 摄取和分解代谢的蛋白质。此外，araE（编码 Ara 转运蛋白）的基因组表达被持续增强。由此产生的菌株被命名为 BAD-5。通过在 Ara 的亚饱和水平下表达更快降解的 GFP（LAA），发现 BAD-5 菌株在有或没有葡萄糖的情况下，80%的细胞肉眼可见亮。通过流式细胞术的进一步分析表明，BAD-5 菌株的 GFP 表达呈均匀分布。相比之下，BL21（BAD）菌株在有葡萄糖存在的情况下，肉眼可见亮的细胞不到细胞群体的 10%，并且仍然是暗的。此外，无论葡萄糖存在与否，BAD-5 菌株在 20 μM Ara 下都可以很容易地获得来自非洲爪蟾（Rluc）的饱和水平的荧光素酶。BL21（BAD）菌株中的 Rluc 以 Ara 剂量依赖的方式产生，并且当存在葡萄糖时，蛋白质产生会停止。总体而言，它说明了改进的系统在高效、均匀且不依赖葡萄糖的情况下用于重组蛋白过量生产的有用性。
