Defining mechanisms of plant-pathogen interactions by quantitative proteomics for improved strategies in molecular farming

重组蛋白在植物中的瞬时表达越来越多地被用作生产单抗、酶和疫苗等治疗性蛋白的平台。这一系统的好处包括降低药物开发成本，快速向市场交付新产品，以及为关节炎和癌症等疾病提供安全有效的药物的能力。PlantForm Corporation是一家加拿大生物技术公司，该公司开发了这样一种系统，以生产生物相似版本的治疗药物(例如Humira®和Herceptin®)，以及针对病原体(例如HIV和埃博拉病毒)的创新产品。PlantForm的技术名为vivoXPRESS®，其基础是使用一种常见的植物病原体(根癌农杆菌)将编码特定蛋白质产品的基因输送到植物细胞(本氏烟草)。这种方法提供了一种技术，在这种技术中，工厂变成了生产所需产品的工厂。最近与E.Allen-Vercoe博士(Guelph大学NSERC CRD项目的合作者)最近获得的NSERC Engage赠款(2017)导致了使用微生物发酵生产农杆菌培养的可扩展方法。然而，尽管取得了这些进展，但从病原体和宿主的角度对农杆菌渗透的影响还没有得到全面的了解。在这里，拟议的研究项目直接建立在NSERC Engage赠款的成功基础上，以分析农业渗入后根癌农杆菌和本底农杆菌中蛋白质和代谢物丰度的差异，以确定生产过程中的步骤，从而为最佳目标蛋白质生产量身定做。这一研究项目的结果将为了解感染过程中病原体和宿主在细胞水平上的调控模式提供有价值的见解，并表征农杆菌传递蛋白质的机制。我们预计，我们的结果将转化为蛋白质产量的大幅增加，并为PlantForm公司的新产品开发节省大量成本。最终，我们的目标是减少加拿大生产新的、安全和有效的治疗药物的障碍。

Transient expression of recombinant proteins in plants is increasingly being used as a production platform for therapeutic proteins such as monoclonal antibodies, enzymes, and vaccines. Benefits of this system include a reduced cost of drug development, rapid delivery of new products to the market, and an ability to provide safe and efficacious medicines for diseases such as arthritis and cancer. PlantForm Corporation is a Canadian biotechnology company that has developed such a system to produce biosimilar versions of therapeutics (e.g. Humira® and Herceptin®), as well as innovative products against pathogens (e.g. HIV and Ebola virus). The basis of PlantForm's technology, known as vivoXPRESS®, is to use a common plant pathogen (Agrobacterium tumefaciens) to deliver genes encoding for a specific protein product to plant cells (Nicotiana benthamiana). This approach provides a technology in which the plant becomes a factory for manufacturing of the desired product. A recent NSERC Engage grant (2017) with Dr. E. Allen-Vercoe (collaborator on this NSERC CRD project; University of Guelph) resulted in scalable methods using microbial fermentation for production of Agrobacterium cultures. However, despite these advances, a comprehensive understanding of the impact of Agrobacterium infiltration from both the pathogen and host perspectives, has not been performed. Here, the proposed research project builds directly upon the success of the NSERC Engage grant to profile differences in protein and metabolite abundance in A. tumefaciens and N. benthamiana following agroinfiltration to determine steps in the production process, which can be tailored for optimal target protein production. The results from this research project will provide valuable insight into patterns of regulation at the cellular level in the pathogen and host during infection and characterize mechanisms of protein delivery by Agrobacterium. We anticipate our results to translate into substantial increases in protein production and significant cost savings for new product development for PlantForm. Ultimately, we aim to reduce barriers for the production of new, safe, and effective therapeutics in Canada.