Institut für Physikalische und Theoretische Chemie, NanoBioSciences, Technische Universität Braunschweig, Hans Sommer Str, 10, Braunschweig 38106, Germany.
Microb Cell Fact. 2013 May 20;12:49. doi: 10.1186/1475-2859-12-49.
In current protein research, a limitation still is the production of active recombinant proteins or native protein associations to assess their function. Especially the localization and analysis of protein-complexes or the identification of modifications and small molecule interaction partners by co-purification experiments requires a controllable expression of affinity- and/or fluorescence tagged variants of a protein of interest in its native cellular background. Advantages of periplasmic and/or homologous expressions can frequently not be realized due to a lack of suitable tools. Instead, experiments are often limited to the heterologous production in one of the few well established expression strains.
Here, we introduce a series of new RK2 based broad host range expression plasmids for inducible production of affinity- and fluorescence tagged proteins in the cytoplasm and periplasm of a wide range of Gram negative hosts which are designed to match the recently suggested modular Standard European Vector Architecture and database. The vectors are equipped with a yellow fluorescent protein variant which is engineered to fold and brightly fluoresce in the bacterial periplasm following Sec-mediated export, as shown from fractionation and imaging studies. Expression of Strep-tag®II and Twin-Strep-tag® fusion proteins in Pseudomonas putida KT2440 is demonstrated for various ORFs.
The broad host range constructs we have produced enable good and controlled expression of affinity tagged protein variants for single-step purification and qualify for complex co-purification experiments. Periplasmic export variants enable production of affinity tagged proteins and generation of fusion proteins with a novel engineered Aequorea-based yellow fluorescent reporter protein variant with activity in the periplasm of the tested Gram-negative model bacteria Pseudomonas putida KT2440 and Escherichia coli K12 for production, localization or co-localization studies. In addition, the new tools facilitate metabolic engineering and yield assessment for cytoplasmic or periplasmic protein production in a number of different expression hosts when yields in one initially selected are insufficient.
在当前的蛋白质研究中，一个限制仍然是生产活性重组蛋白或天然蛋白复合物，以评估它们的功能。特别是通过共纯化实验对蛋白质复合物的定位和分析，或者鉴定修饰和小分子相互作用伙伴，需要可控地表达亲和标记和/或荧光标记的目的蛋白的变体，在其天然细胞背景下。由于缺乏合适的工具，周质和/或同源表达的优势通常无法实现。相反，实验通常限于少数几种已建立的表达菌株中的异源生产。
在这里，我们引入了一系列基于 RK2 的新的广谱宿主表达质粒，用于在广泛的革兰氏阴性宿主的细胞质和周质中诱导生产亲和标记和荧光标记的蛋白质，这些质粒旨在与最近提出的模块化标准欧洲载体架构和数据库匹配。这些载体配备了一个黄色荧光蛋白变体，该变体经过 Sec 介导的输出后在细菌周质中折叠并发出明亮的荧光，这从分馏和成像研究中得到了证明。在 Pseudomonas putida KT2440 中，展示了各种 ORF 上 Strep-tag®II 和 Twin-Strep-tag®融合蛋白的表达。
我们生产的广谱宿主构建体能够很好地控制亲和标记蛋白变体的表达，用于单步纯化，并适合复杂的共纯化实验。周质输出变体能够生产亲和标记蛋白，并生成融合蛋白与一种新型工程化的基于 Aequorea 的黄色荧光报告蛋白变体，该变体在测试的革兰氏阴性模型菌 Pseudomonas putida KT2440 和 Escherichia coli K12 的周质中具有活性，用于生产、定位或共定位研究。此外，当最初选择的一种表达宿主中的产量不足时，新工具还可以促进细胞质或周质蛋白生产的代谢工程和产率评估。
