Abstract
Cynara cardunculus suspension cells were transformed by particle bombardment to overexpress the cypro11 gene coding for cyprosin B. Green fluorescent protein, used as a visual reporter through mgfp4-ER gene, facilitates the screening of transformed cells at the initial stages when antibiotics cause generalized cell death. mgfp4-ER lacks a cryptic intron and has an endoplasmic reticulum target sequence, these traits conferring an adequate use as screenable marker for transformed cells. Selected transformed cells, grown in a bioreactor, produced 3.8 g dcw l−1 of biomass, 80 mg l−1 of total protein and 2,060 U ml−1 of enzymatic activity. Specific activity of cyprosin B, purified by anionic-exchange chromatography, was 215 U mg−1 with a purification degree of 8.3-fold. The cyprosin B activity is optimal at 42°C for pH 5.1 and is inhibited by pepstatin A. The results encourage the overexpression of cypro11 gene in transformed C. cardunculus cells leading to high yields of cyprosin B production in bioreactor, which can be considered adequate for industrial production.
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Abbreviations
- BA:
-
Benzyladenine
- CaMV:
-
Cauliflower mosaic virus
- CTAB:
-
Cetyltrimethylammonium bromide
- DEAE:
-
Diethylamino-ethyl
- 2,4-D:
-
2,4-Dichlorophenoxy acetic acid
- ER:
-
Endoplasmic reticulum
- FITC:
-
Fluorescein isothiocyanate
- GFP:
-
Green fluorescence protein
- hph :
-
Hygromycin phosphotransferase gene
- nptII :
-
Neomycin phosphotransferase II gene
- PMSF:
-
Phenylmethylsulphonyl fluoride
- KLA:
-
Oxygen transport coefficient
- VVM:
-
Bioreactor volume per minute
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Acknowledgments
The authors wish to thank Dr. M. Pietrzak for providing the cypro11 gene. We also thank G. Jach for supplying the constructs used for co-transformation experiments (pCATgfp, p35Sm-gfp4-ER). The first author also acknowledges the Foundation for Science and Technology, Portugal, for financial support under the PhD grant no SFRH/BD/8780/2002.
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Sampaio, P.N., Neto, H., Poejo, P. et al. Overexpression and characterization of cyprosin B in transformed suspension cells of Cynara cardunculus . Plant Cell Tiss Organ Cult 101, 311–321 (2010). https://doi.org/10.1007/s11240-010-9690-z
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DOI: https://doi.org/10.1007/s11240-010-9690-z