RESUMEN
KEY MESSAGE: We successfully developed a method for metabolic isotope labeling of recombinant proteins produced in transgenic tobacco. This enabled assessment of structural integrity of plant-derived therapeutic antibodies by NMR analysis. A variety of expression vehicles have been developed for the production of promising biologics, including plants, fungi, bacteria, insects, and mammals. Glycoprotein biologics often experience altered folding and post-translational modifications that are typified by variant glycosylation patterns. These differences can dramatically affect their efficacy, as exemplified by therapeutic antibodies. However, it is generally difficult to validate the structural integrity of biologics produced using different expression vehicles. To address this issue, we have developed and applied a stable-isotope-assisted nuclear magnetic resonance (NMR) spectroscopy method for the conformational characterization of recombinant antibodies produced in plants. Nicotiana benthamiana used as a vehicle for the production of recombinant immunoglobulin G (IgG) was grown in a (15)N-enriched plant growth medium. The Fc fragment derived from the (15)N-labeled antibody thus prepared was subjected to heteronuclear two-dimensional (2D) NMR measurements. This approach enabled assessment of the structural integrity of the plant-derived therapeutic antibodies by comparing their NMR spectral properties with those of an authentic IgG-Fc derived from mammalian cells.
Asunto(s)
Nicotiana/genética , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Adalimumab/genética , Secuencia de Aminoácidos , Secuencia de Carbohidratos , Glicosilación , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/aislamiento & purificación , Inmunoglobulina G/química , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Datos de Secuencia Molecular , Isótopos de Nitrógeno , Plantas Modificadas Genéticamente/genética , Proteínas Recombinantes/metabolismo , Reproducibilidad de los Resultados , Nicotiana/metabolismoRESUMEN
The development of functional protein production systems using transgenic plants as hosts has been rapidly progressing in recent years. Lettuce (Lactuca sativa L.) has been studied as one such host, and it has been reported that the biomass of lettuce per area and target protein expression level can be increased by optimizing the cultivation conditions. Therefore, we investigated methods to minimize the input light energy per target protein to reduce production costs. Herein, we examined the yield of a nontoxic B subunit of Stx2e (Stx2eB) from transgenic lettuce under various cultivation conditions. Stx2eB acts as a vaccine against swine edema disease. The effects of photon flux densities (PPFDs), photoperiod, and light source on Stx2eB production were examined and the findings suggested that 400 µmol m-2 s-1, 24 h, and white LED lamps, respectively, contributed to energy-efficient Stx2eB production. In addition, Stx2eB was produced 1.4 times more efficiently per unit area time using a high plant density (228.5 plants m-2) than a common density (30.4 plants m-2). The findings of the present study can facilitate the development of energy-efficient and low-cost production processes for vaccine protein production, considering temporal and spatial perspectives.
Asunto(s)
Edematosis Porcina , Animales , Edema , Lactuca/metabolismo , Fotoperiodo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , PorcinosRESUMEN
Linoleic acid (18:2n-6) and alpha-linolenic acid (18:3n-3) are polyunsaturated fatty acids that are essential for mammalian nutrition, because mammals lack the desaturases required for synthesis of Delta12 (n-6) and n-3 fatty acids. Many plants can synthesize these fatty acids and, therefore, to examine the effects of a plant desaturase in mammals, we generated transgenic pigs that carried the fatty acid desaturation 2 gene for a Delta12 fatty acid desaturase from spinach. Levels of linoleic acid (18:2n-6) in adipocytes that had differentiated in vitro from cells derived from the transgenic pigs were approximately 10 times higher than those from wild-type pigs. In addition, the white adipose tissue of transgenic pigs contained approximately 20% more linoleic acid (18:2n-6) than that of wild-type pigs. These results demonstrate the functional expression of a plant gene for a fatty acid desaturase in mammals, opening up the possibility of modifying the fatty acid composition of products from domestic animals by transgenic technology, using plant genes for fatty acid desaturases.