Expression analysis of human β-secretase in transgenic tomato fruits

• Published in: Protein expression and purification 2012-03, Vol.82 (1), p.125-131
• Main Authors: Kim, H.-S., Youm, J.-W., Moon, K.-B., Ha, J.-H., Kim, Y.-H., Joung, H., Jeon, J.-H.
• Format: Article
• Language: English
• Subjects: Acidity; Alzheimer Disease - enzymology; Alzheimer Disease - immunology; Alzheimer Disease - prevention & control; Alzheimer Vaccines - genetics; Alzheimer's disease; Alzheimer’s vaccine; Amyloid Precursor Protein Secretases - genetics; Amyloid Precursor Protein Secretases - isolation & purification; Amyloid Precursor Protein Secretases - metabolism; Aspartic Acid Endopeptidases - genetics; Aspartic Acid Endopeptidases - isolation & purification; Aspartic Acid Endopeptidases - metabolism; beta -Site APP cleaving enzyme 1; Boiling; Enzymes; Escherichia; Fluorescence; Fruit - genetics; Fruits; Gene Expression; Genetic Vectors; Heat; Humans; Immune response; Immunosuppressive agents; Lycopersicon esculentum; Lycopersicon esculentum - genetics; Neurodegenerative diseases; Plants, Genetically Modified - genetics; protein purification; Proteolysis; Recombinant protein; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Secretase; Temperature effects; Transgenic plant; Transgenic plants; Vaccines; Western blotting; β-Secretase
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2011.11.012

► Human β-secretase was successfully expressed in transgenic tomato fruit. ► We estimated the expression level of rBACE1 protein at 136±7ngmg−1 TSP. ► Tomato-derived rBACE1 retains its activity for a long storage period. ► Tomato-derived rBACE is severely degraded by heat or boiling. ► The proteolytic activity was similar to that of a commercial sample of Escherichia coli-derived BACE1. An emerging strategy in biomanufacturing involves using transgenic plants to express recombinant pharmaceutical and industrial proteins in large quantities. β-Site APP cleaving enzyme 1 (β-secretase 1, BACE1) is an enzyme involved in the abnormal production of Aβ42, the major component of senile plaques in Alzheimer’s disease (AD). Thus, BACE1 represents a key target protein in the development of new potential drugs to treat Alzheimer’s disease. We aimed to develop a tomato-derived recombinant BACE1 (rBACE1) protein to serve as a vaccine antigen that would promote an immune response. We utilized a plant expression cassette, pE8BACE, to optimize BACE1 expression in tomato fruits. Polyemerase chain reaction and Southern blot analyses verified integration of the BACE1 gene into the plant genome. Northern and Western blot analyses demonstrated successful mRNA and protein expression of rBACE1, respectively; the Sensizyme assay kit estimated the expression level of rBACE1 protein at 136±7ngmg−1 total soluble protein. The tomato-derived rBACE1 retains its activity for a long storage period at cool or room temperature, and is highly resistant to degradation in conditions such as low acidity. Tomato-derived rBACE1 was severely degraded by heat or boiling. The proteolytic activity of tomato-derived rBACE1, confirmed by fluorescence resonance transfer assay, was similar to that of a commercial sample of Escherichia coli-derived BACE1.