International Center for Biotechnology, Osaka University, Suita-shi, Osaka 565-0871, Japan.
Int J Mol Sci. 2018 Jul 6;19(7):1972. doi: 10.3390/ijms19071972.
Gaucher disease is caused by a deficiency of the enzyme glucocerebrosidase (GCase). Currently, enzyme-replacement therapy using recombinant GCase produced in mammalian cells is considered the most effective treatment. Plants are an attractive alternative host for recombinant protein production due to the low cost of large-scale production and lack of risk of contamination by human pathogens. Compared to whole plants, root cultures can grow faster. Therefore, this study aimed to produce recombinant GCase in a root culture. Root culture of a GCase-producing transgenic plant was induced by indole-3-acetic acid at the concentration of 1 mg/L. Recombinant GCase was successfully produced in roots as a functional protein with an enzyme activity equal to 81.40 ± 17.99 units/mg total protein. Crude proteins were extracted from the roots. Recombinant GCase could be purified by concanavalin A and phenyl 650C chromatography. The productivity of GCase was approximately 1 µg/g of the root. A -glycan analysis of purified GCase was performed using nano LC/MS. The Man₃XylFucGlcNAc₂ structure was predominant in purified GCase with two plant-specific glycan residues. This study presents evidence for a new, safe and efficient system of recombinant GCase production that might be applied to other recombinant proteins.
戈谢病是由葡糖脑苷脂酶（GCase）缺乏引起的。目前，使用在哺乳动物细胞中产生的重组 GCase 的酶替代疗法被认为是最有效的治疗方法。由于大规模生产的成本低且不存在人类病原体污染的风险，植物成为重组蛋白生产的有吸引力的替代宿主。与整个植物相比，根培养物可以更快地生长。因此，本研究旨在根培养物中生产重组 GCase。用 1mg/L 的吲哚-3-乙酸诱导产生 GCase 的转基因植物的根培养物。重组 GCase 作为一种具有等于 81.40±17.99 单位/mg 总蛋白的酶活性的功能性蛋白成功地在根中产生。从根中提取粗蛋白。通过伴刀豆球蛋白 A 和苯基 650C 色谱可纯化重组 GCase。GCase 的生产率约为每克根 1µg。使用纳升 LC/MS 对纯化的 GCase 进行 -聚糖分析。在纯化的 GCase 中，主要存在 Man₃XylFucGlcNAc₂结构，具有两个植物特异性聚糖残基。本研究为重组 GCase 生产提供了新的、安全且高效的系统证据，该系统可能适用于其他重组蛋白。
