Plant vesicular transport engineering-molecular mechanisms of selective vesicular transport and application for recombinant protein production in plants

植物囊泡转运工程-选择性囊泡转运的分子机制及其在植物重组蛋白生产中的应用

基本信息

批准号：
14350436
负责人：
YOSHIDA Kazuya
金额：
$ 9.02万
依托单位：
Nara Institute of Science and Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

Peroxidases (PRX, EC 1.11.1.7) are widely distributed across microorganisms, plants, and animals, and in plants have been implicated in a variety of secondary metabolic reactions. In particular, horseradish (Armoracia rusticana) root represents the main source of commercial PRX production. The prxC1a gene, which encodes horseradish PRX (HRP) C, is expressed mainly in the roots and stems of the horseradish plant. HRP C1a protein was shown to be synthesized as a preprotein wish both an N-terminal (NTPP) and a C-terminal propeptide (CTPP). These propeptides, which might be responsible for intracellular localization or secretion, are removed before or concomitant with production of the mature protein. We investigated the functional role of HRP C1a NTPP and CTPP in the determination of the vesicular transport route, with using analytic system of transgenic cultured tobacco cells (Nicotiana tabacum, BY2).In this research, we found out that NTPP and CTPP are necessary and sufficient for accurate localization of mature HRP C1a protein to vacuoles of the vesicular transport system. HRP C1a could be secreted from cells expressing HRP C1a gene without the DNA region encoding the CTPP. We also investigated the roles of the NTPP and the CTPP of HRP C1a in the tobacco plant. In transgenic tobacco plants expressing the HRP C1a gene with NTPP and CTPP,HRP C1a was accumulated in the vacuole. When HRP C1a lacking the CTPP was expressed, active enzyme was secreted into the apoplast space. And, we could enhance the expression of HRP C1a by the use of translational enhancer, NtADH 5'-UTR. Furthermore, we determined the glycan structure of recombinant C1a protein in BY2 cells was Man3FucXylGlcNAc which was same structure as that of endogenous vacuolar glycol-proteins.

过氧化物酶（PRX，EC 1.11.1.7）广泛分布在微生物，植物和动物之间，并且在植物中与多种二级代谢反应有关。特别是，辣根（Armoracia Rusticana）根代表了商业PRX生产的主要来源。编码辣根PRX（HRP）C的PRXC1A基因主要在辣根植物的根和茎上表达。 HRP C1a蛋白被证明是合成的，是N端（NTPP）和C末端丙肽（CTPP）的预蛋白愿望。这些可能负责细胞内定位或分泌的丙肽在成熟蛋白的产生之前被去除或伴随着。 We investigated the functional role of HRP C1a NTPP and CTPP in the determination of the vesicular transport route, with using analytic system of transgenic cultured tobacco cells (Nicotiana tabacum, BY2).In this research, we found out that NTPP and CTPP are necessary and sufficient for accurate localization of mature HRP C1a protein to vacuoles of the vesicular transport system.可以从表达HRP C1a基因的细胞中分泌HRP C1a，而无需编码CTPP的DNA区域。我们还研究了HRP C1A在烟草植物中的NTPP和CTPP的作用。在用NTPP和CTPP表达HRP C1A基因的转基因烟草植物中，液泡中积累了HRP C1A。当表达缺乏CTPP的HRP C1A时，将活性酶分泌到凋亡空间中。而且，我们可以通过使用翻译增强剂Ntadh 5'-UTR来增强HRP C1A的表达。此外，我们确定了BY2细胞中重组C1a蛋白的聚糖结构是MAN3FucxylGlCNAC，其结构与内源性液泡甘油糖蛋白的结构相同。