Production and secretion of recombinant thaumatin in tobacco hairy root cultures

• Published in: Biotechnology journal 2012-04, Vol.7 (4), p.537-545
• Main Authors: Pham, Ngoc Bich, Schäfer, Holger, Wink, Michael
• Format: Article
• Language: English
• Subjects: Agrobacterium rhizogenes; Endoplasmic Reticulum - drug effects; Endoplasmic Reticulum - metabolism; Hairy roots; Nicotiana - genetics; Nicotiana - metabolism; Plant Proteins - genetics; Plant Proteins - metabolism; Plant Roots - drug effects; Plant Roots - genetics; Plant Roots - metabolism; Plants, Genetically Modified - drug effects; Plants, Genetically Modified - genetics; Plants, Genetically Modified - metabolism; Povidone - pharmacology; Recombinant protein; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sodium Chloride - pharmacology; Sweet-tasting protein; Thaumatin
• ISSN: 1860-6768; 1860-7314
• DOI: 10.1002/biot.201100430

Production of recombinant proteins in plant cell or organ cultures and their secretion into the plant cell culture medium simplify the purification procedure and increase protein yield. In this study, the sweet‐tasting protein thaumatin I was expressed and successfully secreted from tobacco hairy root cultures. The presence of an ER signal peptide appears to be crucial for the secretion of thaumatin: without an ER signal peptide, no thaumatin was detectable in the spent medium, whereas inclusion of the ER signal peptide calreticulin fused to the N terminus of thaumatin led to the secretion of thaumatin into the spent medium of hairy root cultures at concentrations of up to 0.21 mg/L. Extracellular thaumatin levels reached a maximum after 30 days (stationary phase) and the subsequent decline was linked to the rapid increase of proteases in the medium. Significant amounts of thaumatin were trapped in the apoplastic space of the root cells. The addition of polyvinylpyrrolidone and sodium chloride into the culture medium led to an increase of extracellular thaumatin amounts up to 1.4 and 2.63 mg/L, respectively. Thaumatin production compares well with yields from other transgenic plants, so that tobacco hairy roots can be considered an alternative production platform of thaumatin. See accompanying commentary by Eva Stoger DOI: 10.1002/biot.201100472 Rhizosecretion of recombinant thaumatin: Tobacco hairy root cultures are a suitable expression platform for the production and secretion of recombinant thaumatin. Thaumatin is a sweet‐tasting protein with high potential to serve as a substitute for sugars or commonly used sweeteners.