A novel T7 RNA polymerase dependent expression system for high-level protein production in the phototrophic bacterium Rhodobacter capsulatus
| Autor: | Achim Heck, Franco Circolone, Vera Svensson, Rene Bergmann, Nadine Katzke, Annette Markert, Thomas Drepper, Karl-Erich Jaeger, Solmaz Arvani |
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| Rok vydání: | 2010 |
| Předmět: |
Yellow fluorescent protein
Molecular Sequence Data macromolecular substances medicine.disease_cause Rhodobacter capsulatus Viral Proteins Bacterial Proteins medicine T7 RNA polymerase Promoter Regions Genetic Escherichia coli Gene Polymerase Gene Regulation of gene expression Rhodobacter Base Sequence biology DNA-Directed RNA Polymerases Gene Expression Regulation Bacterial biology.organism_classification Molecular biology Luminescent Proteins Biochemistry biology.protein Heterologous expression Biotechnology medicine.drug |
| Zdroj: | Protein Expression and Purification. 69:137-146 |
| ISSN: | 1046-5928 |
| Popis: | The functional expression of heterologous genes using standard bacterial expression hosts such as Escherichia coli is often limited, e.g. by incorrect folding, assembly or targeting of recombinant proteins. Consequently, alternative bacterial expression systems have to be developed to provide novel strategies for protein synthesis exceeding the repertoire of the standard expression host E. coli. Here, we report on the construction of a novel expression system that combines the high processivity of T7 RNA polymerase with the unique physiological properties of the facultative photosynthetic bacterium Rhodobacter capsulatus. This system basically consists of a recombinant R. capsulatus T7 expression strain (R. capsulatus B10S-T7) harboring the respective polymerase gene under control of a fructose inducible promoter. In addition, a set of different broad-host-range vectors (pRho) was constructed allowing T7 RNA polymerase dependent and independent target gene expression in R. capsulatus and other Gram-negative bacteria. The expression efficiency of the novel system was studied in R. capsulatus and E. coli using the yellow fluorescent protein (YFP) as model protein. Expression levels were comparable in both expression hosts and yielded up to 80mg/l YFP in phototrophically grown R. capsulatus cultures. This result clearly indicates that the novel R. capsulatus-based expression system is well suited for the high-level expression of soluble proteins. |
| Databáze: | OpenAIRE |
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