Dataset Information

Development of an optimized tetracycline-inducible expression system to increase the accumulation of interleukin-10 in tobacco BY-2 suspension cells.

ABSTRACT:

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.

SUBMITTER: Bortesi L

PROVIDER: S-EPMC3410776 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Publications

Development of an optimized tetracycline-inducible expression system to increase the accumulation of interleukin-10 in tobacco BY-2 suspension cells.

Bortesi Luisa L Rademacher Thomas T Schiermeyer Andreas A Schuster Flora F Pezzotti Mario M Schillberg Stefan S

BMC biotechnology 20120711

<h4>Background</h4>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 interleuk ...[more]

PMID: 22784336

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Project description:Blunt trauma and traumatic spinal cord injury induce systemic inflammation that contributes to morbidity. Dysregulated neural control of systemic inflammation postinjury is likely exaggerated in patients with traumatic spinal cord injury. We used in silico methods to discern dynamic inflammatory networks that could distinguish systemic inflammation in traumatic spinal cord injury from blunt trauma.Retrospective study.Tertiary care institution.Twenty-one severely injured thoracocervical traumatic spinal cord injury patients and matched 21 severely injured blunt trauma patients without spinal cord injury.None.Serial blood samples were obtained from days 1 to 14 postinjury. Twenty-four plasma inflammatory mediators were quantified. Statistical significance between the two groups was determined by two-way analysis of variance. Dynamic Bayesian network inference was used to suggest dynamic connectivity and central inflammatory mediators. Circulating interleukin-10 was significantly elevated in thoracocervical traumatic spinal cord injury group versus non-spinal cord injury group, whereas interleukin-1?, soluble interleukin-2 receptor-?, interleukin-4, interleukin-5, interleukin-7, interleukin-13, interleukin-17, macrophage inflammatory protein 1? and 1?, granulocyte-macrophage colony-stimulating factor, and interferon-? were significantly reduced in traumatic spinal cord injury group versus non-spinal cord injury group. Dynamic Bayesian network suggested that post-spinal cord injury interleukin-10 is driven by inducible protein-10, whereas monocyte chemotactic protein-1 was central in non-spinal cord injury dynamic networks. In a separate validation cohorts of 356 patients without spinal cord injury and 85 traumatic spinal cord injury patients, individuals with plasma inducible protein-10 levels more than or equal to 730 pg/mL had significantly prolonged hospital and ICU stay and days on mechanical ventilator versus patients with plasma inducible protein-10 level less than 730 pg/mL.This is the first study to compare the dynamic systemic inflammatory responses of traumatic spinal cord injury patients versus patients without spinal cord injury, suggesting a key role for inducible protein-10 in driving systemic interleukin-10 and morbidity and highlighting the potential utility of in silico tools to identify key inflammatory drivers.

Project description:PurposeIn the programming of tumor-targeting bacteria, various therapeutic or reporter genes are expressed by different gene-triggering strategies. Previously, we engineered pJL87 plasmid with an inducible bacterial drug delivery system that simultaneously co-expressed two genes for therapy and imaging by a bidirectional tet promoter system only in response to the administration of exogenous doxycycline (Doxy). In this multi-cassette expression approach, tetA promoter (PtetA) was 100-fold higher in expression strength than tetR promoter (PtetR). In the present study, we developed pJH18 plasmid with novel Doxy-inducible gene expression system based on a tet promoter.ProceduresIn this system, Tet repressor (TetR) expressed by a weak constitutive promoter binds to tetO operator, resulting in the tight repression of gene expressions by PtetA and PtetR, and Doxy releases TetR from tetO to de-repress PtetA and PtetR.ResultsIn Salmonella transformed with pJH18, the expression balance of bidirectional tet promoters in pJH18 was remarkably improved (PtetA:PtetR = 4~6:1) compared with that of pJL87 (PtetA:PtetR = 100:1) in the presence of Doxy. Also, the expression level by novel tet system was much higher in Salmonella transformed with pJH18 than in those with pJL87 (80-fold in rluc8 and 5-fold in clyA). Interestingly, pJH18 of the transformed Salmonella was much more stably maintained than pJL87 in antibiotic-free tumor-bearing mice (about 41-fold), because only pJH18 carries bom sequence with an essential role in preventing the plasmid-free population of programmed Salmonella from undergoing cell division.ConclusionsOverall, doxycycline-induced co-expression of two proteins at similar expression levels, we exploited bioluminescence reporter proteins with preclinical but no clinical utility. Future validation with clinically compatible reporter systems, for example, suitable for radionuclide imaging, is necessary to develop this system further towards potential clinical application.

Dataset Information

Development of an optimized tetracycline-inducible expression system to increase the accumulation of interleukin-10 in tobacco BY-2 suspension cells.

Background

Results

Conclusions

Publications

Development of an optimized tetracycline-inducible expression system to increase the accumulation of interleukin-10 in tobacco BY-2 suspension cells.

Similar Datasets

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