Broad host range vectors for expression of proteins with (Twin-) Strep- tag, His-tag and engineered, export optimized yellow fluorescent protein

Microbial Cell Factories

Research

Kenneth N Timmis¹  Philip Tinnefeld²  Thorben Dammeyer³ 

[1] Environmental Microbiology Laboratory, Helmholtz Centre for Infection Research, Inhoffenstr. 7, 38124, Braunschweig, Germany;Institut für Mikrobiologie, Technische Universität Braunschweig, Spielmannstr. 7, 38106, Braunschweig, Germany;Institut für Physikalische und Theoretische Chemie, NanoBioSciences, Technische Universität Braunschweig, Hans Sommer Str. 10, 38106, Braunschweig, Germany;Institut für Physikalische und Theoretische Chemie, NanoBioSciences, Technische Universität Braunschweig, Hans Sommer Str. 10, 38106, Braunschweig, Germany;Environmental Microbiology Laboratory, Helmholtz Centre for Infection Research, Inhoffenstr. 7, 38124, Braunschweig, Germany;

关键词: Yellow Fluorescent Protein;    Multiple Cloning Site;    Periplasmic Space;    Broad Host Range;    scFv Antibody;   

DOI  :  10.1186/1475-2859-12-49

 received in 2013-03-06, accepted in 2013-05-09,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundIn 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.ResultsHere, 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.ConclusionThe 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.

【 授权许可】

CC BY   
© Dammeyer et al.; licensee BioMed Central Ltd. 2013