Improvement of glycosyltransferases for in vitro glycan remodeling of plant derived therapeutic proteins.

植物是哺乳动物细胞培养的一种极具成本效益的替代方案，可用于生产以蛋白质为基础的药物，即生物制品。这些药物是从正常人类生物学中提取的蛋白质，它们保护我们免受各种天然和人造毒素、传染病和免疫系统故障(如癌症)的伤害。这些都是以重组蛋白的形式生产的，在组织培养中生产非常昂贵。正常的人类治疗蛋白被称为糖蛋白的分子装饰，这些糖蛋白对人体的稳定性和长寿是必不可少的。植物和动物制造糖蛋白的生化机制几乎相同，导致植物能够制造一些重要的人类蛋白质，如抗体，以及保护人体免受神经毒气等毒素伤害的蛋白质。然而，植物不会自然地在植物上制造人类风格的多糖装饰，而且植物多糖会引起过敏反应。植物工程的最新进展现在允许研究人员在没有植物特有的装饰的情况下生产蛋白质，这使蛋白质成为人类免疫系统的目标。然而，在哺乳动物和植物表达系统中，生产真正的人类多糖装饰物仍然很难控制。PlantForm和瓦卡丘克实验室正在合作，在实验室使用酶来完成与正常人类蛋白质上发现的完全相同的糖链修饰，目标是对目标蛋白质进行商业规模的糖链修饰。为此，通过我们独特的细菌表达系统，正在设计改变多糖所需的酶，以提高产量、活性和稳定性。我们正在开发固定化酶来生产各种末端糖链装饰物，这样蛋白质在反应后不需要大量的重新纯化。我们还在一种不产生有毒污染物--内毒素--的细菌中生产这些酶，这需要付出巨大的努力才能从这些高价值的蛋白质中去除。

Plants represent an excellent cost-effective alternative to mammalian cell culture for production of protein based drugs known as biologics. These drugs are proteins derived from normal human biology, where they protect us from various natural and man-made toxins, infectious disease, and immune system malfunctions like cancer. These are produced as recombinant proteins and are very expensive to manufacture in tissue culture. Normal human therapeutic proteins are decorated with molecules called glycans making them "glycoproteins", and these glycans are essential for stability and longevity in the body. The biochemical machinery for making glycoproteins in plants and animals is almost identical, resulting in plants being able to make some important human proteins, like antibodies, and proteins which protect from toxins like nerve gas. Plants however don't naturally make human style glycan decorations on plants, and plant glycans can cause an allergic reaction. Recent advances in plant engineering now allows researchers to produce proteins without the plant specific decorations which make the proteins a target of the human immune system. However, the production of the authentic human glycan decorations is still difficult to control in both mammalian- and plant-based expression systems. PlantForm, and the Wakarchuk lab are collaborating to use enzymes in the laboratory to finish the glycan decorations exactly as those found on the normal human protein with the goal of commercial-scale glycan modification of target proteins. To this end, the enzymes required to change the glycans are being engineered for improved production, activity and stability through our unique bacterial expression systems. We are developing immobilized enzymes to produce various terminal glycan decorations such that the proteins don't require extensive re-purification after the reactions. We are also engineering production of these enzymes in a bacterium which doesn't produce a toxic contaminant - endotoxin - which requires significant effort to remove from these high value proteins.