Abstract
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.
Highlights
In current protein research, a limitation still is the production of active recombinant proteins or native protein associations to assess their function
To achieve a broad host range, we base our system on a plasmid chassis equipped with the well- established RK2 origin of replication [5,20] that has been demonstrated to be efficiently maintained and replicates in more than 30 Gram negative bacteria [5]
Applicability to a large set of hosts is further increased by an oriT origin of transfer which can be used to transfer the plasmids to a host expression strain that is inaccessible to transformation by allowing for conjugative mobilization of the plasmids
Summary
A limitation still is the production of active recombinant proteins or native protein associations to assess their function. The vast majority of the microbial diversity remains unexplored with regard to experiments involving recombinant production of proteins, due to the limited availability of customized expression tools. This deficit becomes especially aggravating in the emerging era of synthetic and systems biology and is reflected by continuous efforts to develop shuttle plasmids and genetic tools with a broad host range [2,3,4,5]. The remaining gap between the gained information on a heterologously expressed protein and its natural behavior can hardly be closed unless a protein can be and controllably expressed in the bacteria from which it originates This is a specific requirement for many experiments like localization and colocalization studies, mutant complementation experiments, chromatin immunoprecipitation or for identification of protein interaction partners
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
