Abstract
BACKGROUND: Transgenic chloroplasts are potential bioreactors for recombinant protein production, especially for achievement of high levels of protein expression and proper folding. Production of therapeutic proteins in leaves provides transgene containment by elimination of reproductive structures. Therefore, in this study, human Insulin like Growth Factor-1 is expressed in transgenic chloroplasts for evaluation of structural identity and function. RESULTS: Expression of the synthetic Insulin like Growth Factor 1 gene (IGF-1s, 60% AT) was observed in transformed E. coli. However, no native IGF-1 gene (IGF-1n, 41% AT) product was detected in the western blots in E. coli. Site-specific integration of the transgenes into the tobacco chloroplast genome was confirmed after transformation using PCR. Southern blot analysis confirmed that the transgenic lines were homoplasmic. The transgenic plant lines had IGF-1s expression levels of 11.3% of total soluble protein (TSP). The IGF-1n plants contained 9.5% TSP as IGF-1n, suggesting that the chloroplast translation machinery is more flexible than E. coli in codon preference and usage. The expression of IGF-1 was increased up to 32% TSP under continuous illumination by the chloroplast light regulatory elements. IgG-Sepharose affinity column chromatographic separation of Z domain containing chloroplast derived IGF-1 protein, single and two dimensional electrophoresis methods and mass spectrometer analysis confirmed the identity of human IGF-1 in transgenic chloroplasts. Two spots analyzed from 2-D focusing/phoresis acrylamide gel showed the correct amino acid sequence of human IGF-1 and the S. aureus Z-tag. Cell proliferation assays in human HU-3 cells demonstrated the biological activity of chloroplast derived IGF-1 even in the presence of the S. aureus Z tag. CONCLUSION: This study demonstrates that the human Insulin like Growth Factor-1 expressed in transgenic chloroplasts is identical to the native protein and is fully functional. The ability to use plant chloroplasts as bioreactors to generate proteins of great economic value that retain their biological activity is an exciting and achievable goal that appears to be within our grasp.
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Claims 7
Claim · model-written summary
Transgenes integrated into the tobacco chloroplast genome.
- Taxon
- tobacco
Nicotiana tabacume0 - Outcome
- Site-specific integration of the transgenes into the tobacco chloroplast genomee0
Not reportedDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Site-specific integration of the transgenes into the tobacco chloroplast genome was confirmed after transformation using PCR”
Claim · model-written summary
Transgenic lines were homoplasmic.
- Taxon
- tobaccoNicotiana tabacume1
- Outcome
- transgenic lines were homoplasmice0
Not reportedDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Southern blot analysis confirmed that the transgenic lines were homoplasmic”
t0“transgenic chloroplasts”
e1“Site-specific integration of the transgenes into the tobacco chloroplast genome was confirmed after transformation using PCR”
Claim · model-written summary
IGF-1s reached 11.3% of soluble protein in transgenic plants.
- Taxon
- tobaccoNicotiana tabacume1
- Gene
- IGF-1se0
- Outcome
- IGF-1s expression levelse0
- Value
- 11.3% of total soluble protein (TSP)e0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“The transgenic plant lines had IGF-1s expression levels of 11.3% of total soluble protein (TSP)”
t0“transgenic chloroplasts”
e1“Site-specific integration of the transgenes into the tobacco chloroplast genome was confirmed after transformation using PCR”
Claim · model-written summary
IGF-1n accounted for 9.5% of soluble protein in transgenic plants.
- Taxon
- tobaccoNicotiana tabacume1
- Gene
- IGF-1ne0
- Outcome
- IGF-1ne0
- Value
- 9.5% TSPe0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“The IGF-1n plants contained 9.5% TSP as IGF-1n”
t0“transgenic chloroplasts”
e1“Site-specific integration of the transgenes into the tobacco chloroplast genome was confirmed after transformation using PCR”
Claim · model-written summary
Continuous illumination with chloroplast light regulatory elements raised IGF-1 expression to 32% TSP.
- Taxon
- tobaccoNicotiana tabacume1
- Treatment
- continuous illuminatione0
- Gene
- IGF-1e0
- Outcome
- expression of IGF-1e0
- Direction
- increasede0
- Value
- 32% TSPe0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“The expression of IGF-1 was increased up to 32% TSP under continuous illumination by the chloroplast light regulatory elements”
t0“transgenic chloroplasts”
e1“Site-specific integration of the transgenes into the tobacco chloroplast genome was confirmed after transformation using PCR”
Claim · model-written summary
Biochemical analysis confirmed human IGF-1 identity in transgenic chloroplasts.
- Taxon
- tobaccoNicotiana tabacume1
- Gene
- human IGF-1e0
- Outcome
- identity of human IGF-1 in transgenic chloroplastse0
Not reportedDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“mass spectrometer analysis confirmed the identity of human IGF-1 in transgenic chloroplasts”
t0“transgenic chloroplasts”
e1“Site-specific integration of the transgenes into the tobacco chloroplast genome was confirmed after transformation using PCR”
Claim · model-written summary
Cell assays found chloroplast-derived IGF-1 biologically active despite the Z tag.
- Taxon
- tobaccoNicotiana tabacume1
- Gene
- IGF-1e0
- Outcome
- biological activity of chloroplast derived IGF-1e0
- Qualifier
- even in the presence of the S. aureus Z tage0
Not reportedDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Cell proliferation assays in human HU-3 cells demonstrated the biological activity of chloroplast derived IGF-1 even in the presence of the S. aureus Z tag”
t0“transgenic chloroplasts”
e1“Site-specific integration of the transgenes into the tobacco chloroplast genome was confirmed after transformation using PCR”
The same questions in other papers
light · molecular: expression · Nicotiana tabacum (tobacco)Positive and negative results
8 papers in the same direction as this paper · 5 in the opposite direction · 1 reporting no difference from an alternative
- negative
- negative
- negative
11 papers more in this group
See all 15 papers →Claims are grouped by organism, treatment and what was measured or targeted. A paper counts as positive when any of its claims in the group is positive.