Modification of a plant-based expression system for therapeutic protein production through genetic modification of endogenous plant N-glycosylation

植物细胞是一种能够为各种应用生产经济和治疗上重要的蛋白质的系统，被认为是比一些现有的宿主(如细菌或酵母)更安全的生产系统。然而，植物不像哺乳动物细胞那样对蛋白质进行修饰，这可能会对它们的安全性和有效性产生深远的影响。该项目旨在对烟草植物(烟草)进行基因改造，以创建一种基于植物的系统，能够完全使感兴趣的蛋白质“人性化”，同时提高最终产品的安全性。我们的目的是证明哺乳动物的糖基化酶B4GALT1、ST6GAL、GNTIV和GNTV可以在植物细胞中表达，并定位于高尔基体的特定脑池。在特定脑池中酶更有效的情况下，例如内侧高尔基体中的GNTIV和GNTV以及跨高尔基体中的ST6GAL和B4GALT1。我们展示了通过与植物细胞质-跨膜-干细胞(CTS)区域的构建融合来修改定位到所需池的策略。这个项目解决了英国生物研究理事会(BBSRC)关于工业生物技术新战略方法的关键研究领域。该项目将提供一个经济上可行的系统，用于在植物模式生物体中生产高价值的可再生能源和治疗药物，作为现有系统的低免疫原性替代方案，现有系统是通过对植物生物学的基本操作开发的。该项目也符合BBSRC的“世界级支持生物科学”的优先事项，因为糖基化是细胞功能的一个关键方面。

Plant cells are a capable system for producing economically and therapeutically important proteins for a variety of applications, and are considered a safer production system than some existing hosts such as bacteria or yeasts. However, plants do not perform modifications to proteins in the same manner as mammalian cells do, which can have far reaching consequences for their safety and effectiveness. This project aims to genetically modify tobacco plants (Nicotiana tabacum) to create a plant-based system capable of fully "humanizing" proteins of interest, whilst improving safety of final product. We aim to demonstrate that the mammalian glycosylation enzymes B4GALT1, ST6GAL, GNTIV and GNTV can be expressed in plant cells, and localise to specific cisternae of the Golgi body. In instances where enzymes are more effective within specific cisternae, such as GNTIV & GNTV in the medial-Golgi and ST6GAL & B4GALT1 in the trans-Golgi. We demonstrate strategies to modify localisation to the desired cisternae by use of construct fusions with plant Cytoplasmic-Transmembrane-Stem (CTS) regions.This project addresses key areas of study for the British Biological Research Council (BBSRC) on new strategic approaches to industrial biotechnology. The project would offer an economically viable system for the production of high value renewables and therapeutics in a plant model organism as a low immunogenic alternative to existing systems, developed by fundamental manipulation of plant biology. This project aligns also with the BBSRC priority of 'World Class Underpinning Bioscience' as glycosylation is a critical aspect of cell functionality.