Production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expressing bacterial PNGase F
Production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expressing bacterial PNGase F
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DOI：
10.1111/j.1467-7652.2012.00694.x
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发表时间：
2012-09-01
影响因子：
13.8
通讯作者：
Yusibov, Vidadi
中科院分区：
文献类型：
--
作者：
Mamedov, Tarlan;Ghosh, Ananya;Yusibov, Vidadi
分子生物学和基因组学工具的应用正日益导致基于重组蛋白的生物制剂的发展。因此，它导致具有重要健康应用的靶标的多样性增加，并且由于复杂的翻译后修饰而需要更灵活的表达方法。例如，疟原虫寄生虫可能具有复杂的后修饰蛋白质，如Pfs 48/45，其不携带N-连接的聚糖（Exp.寄生醇1998年; 90，165。）但含有在哺乳动物和植物系统中表达期间可能异常糖基化的潜在N-连接糖基化位点。因此，重要的是开发用于产生这些靶标的非糖基化形式以保持生物活性和天然构象的策略。在这项研究中，我们描述了重组N-糖基化蛋白的体内去糖基化，作为其与细菌PNGase F（肽：N-糖苷酶F）瞬时共表达的结果。此外，我们表明，在体内去糖基化的植物产生的疟疾疫苗候选人，Pfs 48 F1，单克隆抗体I，III和V提出的各种表位（I，III和V）的原生恶性疟原虫Pfs 48/45的识别，是显着更强的糖基化形式的植物产生的Pfs 48 F1相比。据我们所知，以前在任何真核系统（包括植物）中都没有实现体内酶促蛋白质去糖基化，也没有在植物系统中表达细菌PNGase F。因此，在这里，我们报告的第一次在植物中表达的活性细菌酶PNGase F和生产的重组蛋白的目的在非糖基化的形式。
Application of tools of molecular biology and genomics is increasingly leading towards the development of recombinant protein-based biologics. As such, it is leading to an increased diversity of targets that have important health applications and require more flexible approaches for expression because of complex post-translational modifications. For example, Plasmodium parasites may have complex post-translationally modified proteins such as Pfs48/45 that do not carry N-linked glycans (Exp. Parasitol. 1998; 90, 165.) but contain potential N-linked glycosylation sites that can be aberrantly glycosylated during expression in mammalian and plant systems. Therefore, it is important to develop strategies for producing non-glycosylated forms of these targets to preserve biological activity and native conformation. In this study, we are describing in vivo deglycosylation of recombinant N-glycosylated proteins as a result of their transient co-expression with bacterial PNGase F (Peptide: N-glycosidase F). In addition, we show that the recognition of an in vivo deglycosylated plant-produced malaria vaccine candidate, Pfs48F1, by monoclonal antibodies I, III and V raised against various epitopes (I, III and V) of native Pfs48/45 of Plasmodium falciparum, was significantly stronger compared to that of the glycosylated form of plant-produced Pfs48F1. To our knowledge, neither in vivo enzymatic protein deglycosylation has been previously achieved in any eukaryotic system, including plants, nor has bacterial PNGase F been expressed in the plant system. Thus, here, we report for the first time the expression in plants of an active bacterial enzyme PNGase F and the production of recombinant proteins of interest in a non-glycosylated form.