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Overexpression and characterization of cyprosin B in transformed suspension cells of Cynara cardunculus

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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Correspondence to Pedro Nuno de Sousa Sampaio.

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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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Keywords

  • Plant cell transformation
  • Bioreactor
  • GFP
  • Cynara cardunculus
  • Cyprosin B