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

• Published in: Plant cell, tissue and organ culture Vol. 101; no. 3; pp. 311 - 321
• Main Authors: Sampaio, Pedro Nuno de Sousa, Neto, Hélia, Poejo, Pedro, Serrazina, Susana Maria T, Pais, Maria Salomé Soares
• Format: Journal Article
• Language: English
• Published: Dordrecht Dordrecht : Springer Netherlands 01.06.2010; Springer Netherlands; Springer; Springer Nature B.V
• Subjects: Antibiotics; aspartic proteinases; biolistics; Biological and medical sciences; Biomedical and Life Sciences; Bioreactors; Biotechnology; Bombardment; cardoons; Cell death; cell suspension culture; Cynara cardunculus; Cyprosin; cyprosin B; dry matter accumulation; Endoplasmic reticulum; Enzymatic activity; enzyme activity; Fluorescence; Fundamental and applied biological sciences. Psychology; gene expression; germplasm screening; Green fluorescent protein; Industrial production; introns; Life Sciences; Lymphocytes B; Methods. Procedures. Technologies; molecular sequence data; optimization; Original Paper; Particle bombardment; Plant Genetics and Genomics; Plant Pathology; Plant Physiology; plant proteins; Plant Sciences; protein transport; Proteins; Transformed cells; transgenic plants; Various methods and equipments; vegetable crops; GFP; Plant cell transformation; Cyprosin B; Bioreactor; Cell culture; Vegetals; Gene overexpression; Compositae; Characterization; Suspension culture; Cynara cardunculus; Dicotyledones; Angiospermae; Green fluorescent protein; Spermatophyta
• ISSN: 0167-6857; 1573-5044
• DOI: 10.1007/s11240-010-9690-z

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.