Expression analysis of the nucleocytoplasmic lectin 'Orysata' from rice in Pichia pastoris
Expression analysis of the nucleocytoplasmic lectin 'Orysata' from rice in Pichia pastoris
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DOI：
10.1111/j.1742-4658.2011.08122.x
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发表时间：
2011-06-01
期刊：
影响因子：
5.4
通讯作者：
Van Damme, Els J. M.
中科院分区：
文献类型：
--
作者：
Al Atalah, Bassam;Fouquaert, Elke;Van Damme, Els J. M.
水稻凝集素（Oryza sativa lectin），缩写为Orysata，是一种甘露糖特异性的、与jacalin相关的凝集素，在暴露于某些胁迫条件后在水稻植物中表达。将水稻凝集素序列与增强型绿色荧光蛋白连接后，在烟草Bright Yellow 2细胞中表达，结果表明Orysata位于植物细胞的细胞核和细胞质中，属于核质jacalin相关凝集素类。由于Orysata在水稻组织中的表达水平很低，因此在具有酵母α因子序列的甲醇营养型酵母Pichia pastoris中表达凝集素，以将重组蛋白引导到分泌途径中并将蛋白表达到培养基中。每升培养基纯化约12 mg重组凝集素。SDS/PAGE和westernblot分析表明，重组凝集素以两种分子形式存在。远蛋白质印迹分析表明，23 kDa的凝集素多肽含有N-聚糖，这是不存在于18.5 kDa的多肽。重组Orysata中存在的聚糖的表征揭示了高甘露糖结构，Man 9 -11聚糖是最丰富的。聚糖阵列分析表明，Orysata与高甘露糖以及更复杂的N-聚糖结构相互作用。Orysata在细胞培养中具有有效的抗人类免疫缺陷病毒和抗呼吸道合胞病毒活性，与其他jacalin相关的凝集素相比。
The Oryza sativa lectin, abbreviated Orysata, is a mannose-specific, jacalin-related lectin expressed in rice plants after exposure to certain stress conditions. Expression of a fusion construct containing the rice lectin sequence linked to enhanced green fluorescent protein in Bright Yellow 2 tobacco cells revealed that Orysata is located in the nucleus and the cytoplasm of the plant cell, indicating that it belongs to the class of nucleocytoplasmic jacalin-related lectins. Since the expression level of Orysata in rice tissues is very low the lectin was expressed in the methylotrophic yeast Pichia pastoris with the Saccharomyces alpha-factor sequence to direct the recombinant protein into the secretory pathway and express the protein into the medium. Approximately 12 mg of recombinant lectin was purified per liter medium. SDS/PAGE and western blot analysis showed that the recombinant lectin exists in two molecular forms. Far western blot analysis revealed that the 23 kDa lectin polypeptide contains an N-glycan which is absent in the 18.5 kDa polypeptide. Characterization of the glycans present in the recombinant Orysata revealed high-mannose structures, Man9-11 glycans being the most abundant. Glycan array analysis showed that Orysata interacts with high-mannose as well as with more complex N-glycan structures. Orysata has potent anti-human immunodeficiency virus and anti-respiratory syncytial virus activity in cell culture compared with other jacalin-related lectins.