Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany.
Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany.
Plasmid. 2020 Nov;112:102540. doi: 10.1016/j.plasmid.2020.102540. Epub 2020 Sep 28.
The Escherichia coli/Corynebacterium glutamicum shuttle vector pEKEx2 is an IPTG-inducible expression vector that has been used successfully for the synthesis of numerous proteins in C. glutamicum. We discovered that the leaky gene expression observed for pEKEx2-derived plasmids relates to reduced functionality of the plasmid-encoded repressor LacI carrying a modified C-terminus, while duplicate DNA sequences in the pEKEx2 backbone contribute to plasmid instability. We constructed the pEKEx2-derivatives pPBEx2 and pPREx2, which harbor a restored lacI gene and which lack the unnecessary duplicate DNA sequences. pPREx2 in addition enables fusion of target genes to a C-terminal Strep-tag II coding region for easy protein detection and purification. In the absence of inducer, the novel vectors exhibit tight gene repression in C. glutamicum, as shown for the secretory production of Fusarium solani pisi cutinase and the cytosolic production of green fluorescent protein and C. glutamicum myo-inositol dehydrogenase. Undesired heterogeneity amongst clones expressing cutinase from pEKEx2 was attributed to the loss of a vector fragment containing the cutinase gene, which likely occurred via homologous recombination of the identical flanking DNA sequences. Such loss was not observed for pPBEx2. Using pPREx2, IolG-Strep was successfully produced and purified to homogeneity by Strep-Tactin affinity chromatography, obtaining 1.5 mg IolG with a specific activity of 27 μmol·min·(mg protein) from 100 mL culture. The tight gene repression in the absence of inducer and the improved plasmid stability make expression vectors pPBEx2/pPREx2 attractive alternatives to the available molecular tools for genetic manipulation and high-level production of recombinant proteins in C. glutamicum.
大肠杆菌/谷氨酸棒杆菌穿梭载体 pEKEx2 是一种 IPTG 诱导型表达载体，已成功用于谷氨酸棒杆菌中许多蛋白质的合成。我们发现，pEKEx2 衍生质粒中观察到的漏表达与携带修饰 C 末端的质粒编码的阻遏物 LacI 的功能降低有关，而 pEKEx2 骨架中的重复 DNA 序列有助于质粒不稳定。我们构建了 pEKEx2 衍生物 pPBEx2 和 pPREx2，它们携带一个恢复的 lacI 基因，并且缺乏不必要的重复 DNA 序列。pPREx2 还可以使靶基因融合到 C 末端 Strep-tag II 编码区，便于蛋白质的检测和纯化。在没有诱导剂的情况下，新型载体在谷氨酸棒杆菌中表现出紧密的基因抑制，如丝状镰刀菌角质酶的分泌生产和绿色荧光蛋白和谷氨酸棒杆菌肌醇脱氢酶的胞质生产所示。从 pEKEx2 表达角质酶的克隆表现出的不必要的异质性归因于载体片段的丢失，该载体片段可能通过相同侧翼 DNA 序列的同源重组发生。在 pPBEx2 中没有观察到这种丢失。使用 pPREx2，成功地通过 Strep-Tactin 亲和层析将 IolG-Strep 进行了生产和纯化为均一性，从 100 mL 培养物中获得了 1.5 mg IolG，比活为 27 μmol·min·(mg 蛋白)。在没有诱导剂的情况下的紧密基因抑制和提高的质粒稳定性使表达载体 pPBEx2/pPREx2 成为遗传操作和谷氨酸棒杆菌中重组蛋白高水平生产的现有分子工具的有吸引力的替代品。
