title: Expression analysis of human β-secretase in transgenic tomato fruits.
abstract: 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 ± 7 ng mg⁻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.
authors.affiliation: Plant Systemic Engineering Research Center, KRIBB, Gwahangno 125, Yuseong-gu, Daejeon 305-806, Republic of Korea.
authors.forename: H-S
authors.initials: HS
authors.lastname: Kim
authors.forename: J-W
authors.initials: JW
authors.lastname: Youm
authors.forename: K-B
authors.initials: KB
authors.lastname: Moon
authors.forename: J-H
authors.initials: JH
authors.lastname: Ha
authors.forename: Y-H
authors.initials: YH
authors.lastname: Kim
authors.forename: H
authors.initials: H
authors.lastname: Joung
authors.forename: J-H
authors.initials: JH
authors.lastname: Jeon
mesh_terms: D000544:Alzheimer Disease / Q000201:enzymology / Q000276:immunology / Q000517:prevention & control; D023582:Alzheimer Vaccines / Q000235:genetics*; D053829:Amyloid Precursor Protein Secretases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D016282:Aspartic Acid Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D005638:Fruit / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D018551:Solanum lycopersicum / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D023582:Alzheimer Vaccines; D011994:Recombinant Proteins; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human