Reference - Detail
|Author||Bortesi L, Rademacher T, Schiermeyer A, Schuster F, Pezzotti M, Schillberg S.|
|Title||Development of an optimized tetracycline-inducible expression system to increase the accumulation of interleukin-10 in tobacco BY-2 suspension cells.|
BACKGROUND:Plant cell suspension cultures can be used for the production of valuable pharmaceutical and industrial proteins. When the recombinant protein is secreted into the culture medium, restricting expression to a defined growth phase can improve both the quality and quantity of the recovered product by minimizing proteolytic activity. Temporal restriction is also useful for recombinant proteins whose constitutive expression affects cell growth and viability, such as viral interleukin-10 (vIL-10).
RESULTS:We have developed a novel, tetracycline-inducible system suitable for tobacco BY-2 suspension cells which increases the yields of vIL-10. The new system is based on a binary vector that is easier to handle than conventional vectors, contains an enhanced inducible promoter and 5'-UTR to improve yields, and incorporates a constitutively-expressed visible marker gene to allow the rapid and straightforward selection of the most promising transformed clones. Stable transformation of BY-2 cells with this vector, without extensive optimization of the induction conditions, led to a 3.5 fold increase in vIL-10 levels compared to constitutive expression in the same host.
CONCLUSIONS:We have developed an effective and straightforward molecular farming platform technology that improves both the quality and the quantity of recombinant proteins produced in plant cells, particularly those whose constitutive expression has a negative impact on plant growth and development. Although we tested the platform using vIL-10 produced in BY-2 cells, it can be applied to other host/product combinations and is also useful for basic research requiring strictly controlled transgene expression.
|MeSH||5' Untranslated Regions Agrobacterium tumefaciens / cytology Agrobacterium tumefaciens / growth & development Caulimovirus / genetics Coculture Techniques Genetic Vectors / genetics Genetic Vectors / metabolism Interleukin-10 / genetics Interleukin-10 / metabolism* Plant Cells / drug effects Plant Cells / metabolism Plants, Genetically Modified / cytology Promoter Regions, Genetic Recombinant Proteins / biosynthesis Recombinant Proteins / genetics Tetracycline / pharmacology* Tobacco / cytology* Trans-Activators / genetics|
|WOS Category||BIOTECHNOLOGY & APPLIED MICROBIOLOGY|
|DNA material||pBluescript SKII+mIL10 (RDB01476)|