Directed evolution of the transcription factor XylS for development of improved expression systems.
Directed evolution of the transcription factor XylS for development of improved expression systems.
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DOI：
10.1111/j.1751-7915.2009.00126.x
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发表时间：
2010-01
影响因子：
5.7
通讯作者：
Valla S
中科院分区：
文献类型：
--
作者：
Vee Aune TE;Bakke I;Drabløs F;Lale R;Brautaset T;Valla S
可诱导的Pm启动子及其同源的正转录调节因子XylS已被证明可用于高密度条件下的重组蛋白生产。在这里，我们报道了XylS的定向进化，导致突变蛋白在大肠杆菌中具有增强的转录刺激能力。利用易出错PCR对xylS进行第一轮诱变，构建了一个包含约43万个克隆的文库，由于报告基因bla（编码β -内酰胺酶）的表达水平与相应宿主细胞的氨苄西林耐受性呈正相关，因此该文库可以有效地筛选Pm刺激表达的文库。在诱导条件下，与野生型相比，14种不同的氨基酸替换分别导致了高达近三倍的表达刺激。这些突变都位于蛋白质的N端一半对应的部分。突变的不同组合导致进一步的刺激，通过使用随机洗牌程序然后进行新一轮筛选获得最佳结果（诱导条件下约10倍的刺激）。相同突变体的非诱导表达水平也增加了，但只有大约四倍。通过对XylS的N端结构域的硅三维建模，观察到进化的突变蛋白包含位于预测结构不同部分的取代，包括一个β -桶，据推测负责效应体结合和一个卷曲的线圈，可能对二聚化很重要。用进化的XylS突变蛋白StEP - 13从Pm中表达的宿主毒性抗体片段scFv - phOx的总产量比野生型XylS高约9倍，这表明转录因子的定向进化可以成为实现高水平重组蛋白生产的重要新工具。
The inducible Pm promoter together with its cognate positive transcription regulator XylS has been shown to be useful for recombinant protein production under high cell density conditions. Here we report directed evolution of XylS resulting in mutant proteins with increased ability to stimulate transcription in Escherichia coli from Pm. A first round of mutagenesis using error‐prone PCR on xylS was used to construct a library consisting of about 430 000 clones, and this library could be efficiently screened with respect to stimulation of expression from Pm due to a positive correlation between the level of expression of the reporter gene, bla (encoding β‐lactamase), and the ampicillin tolerance of the corresponding host cells. Fourteen different amino acid substitutions in XylS were found to separately lead to up to nearly a threefold stimulation of expression under induced conditions, relative to wild type. These mutations were all located in the part corresponding to the N‐terminal half of the protein. Varying combinations of the mutations resulted in further stimulation, and the best results (about 10‐fold stimulation under induced conditions) were obtained by using a random shuffling procedure followed by a new round of screening. The uninduced levels of expression for the same mutants also increased, but only about four times. Through in silico 3D modelling of the N‐terminal domain of XylS, it was observed that the evolved mutant proteins contained substitutions that were positioned in different parts of the predicted structure, including a β‐barrel putatively responsible for effector binding and a coiled coil probably important for dimerization. The total production of the host‐toxic antibody fragment scFv‐phOx expressed from Pm with the evolved XylS mutant protein StEP‐13 was about ninefold higher than with wild‐type XylS, demonstrating that directed evolution of transcription factors can be an important new tool to achieve high‐level recombinant protein production.
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影响因子：
17.3
作者：
Hannig, G;Makrides, SC
通讯作者：
Makrides, SC
影响因子：
8
作者：
COLOVOS, C;YEATES, TO
通讯作者：
YEATES, TO
影响因子：
4.4
作者：
Bakke, Ingrid;Berg, Laila;Valla, Svein
通讯作者：
Valla, Svein
影响因子：
3.2
作者：
Haldimann, A;Daniels, LL;Wanner, BL
通讯作者：
Wanner, BL
影响因子：
3.2
作者：
KESSLER, B;HERRERO, M;DELORENZO, V
通讯作者：
DELORENZO, V