Collaborative research: Development of an integrated eukaryotic algae platform for difficult-to-produce therapeutic molecules

可扩展和低成本的生产平台对于开发价格合理的重组蛋白分子越来越重要。在复杂重组蛋白的情况下尤其如此，这些蛋白在商业蛋白质生产平台（如哺乳动物细胞培养和大肠杆菌）中生产通常是困难和昂贵的。由于严格的营养和生长要求、专门的生物反应器设计和环境条件，加上通常的低表达水平，哺乳动物细胞培养生产蛋白质仍然是昂贵的。大肠杆菌虽然能够廉价地生产大量蛋白质，但在生产适当折叠的复杂蛋白质作为可溶性分子方面通常效率低下。莱茵衣藻（Chlamydomonas reinhardtii）是一种单细胞真核藻类，它的生长要求很简单（光、二氧化碳和盐），它作为复杂重组蛋白的生产平台，结合了发酵和细胞培养的许多最有利的特点。莱茵草叶绿体表达的具体特征包括：快速生成稳定转化的主细胞系，在非还原环境中有效折叠哺乳动物蛋白质，宿主代谢耐受“难以表达”的蛋白质，低倾向于不良的翻译后修饰和不溶性聚集体的形成，使用完全确定的无蛋白质无机培养基，细胞在10小时内翻倍，证明了可扩展性，没有内毒素和哺乳动物病毒污染。我们已经证明C. reinhardtii能够产生复杂的多结构域蛋白，这些蛋白不能在其他异种系统中有效表达和纯化。该提案的重点是表达和纯化两种独特且具有重要治疗意义的重组蛋白，这将有助于证明该平台的实用性，并且如果它们能够以具有竞争力的成本生产和纯化，则可能具有高影响。为了将C. reinhardtii开发为生产一类独特的重组蛋白的广泛和有竞争力的平台，具有互补专业知识的跨学科团队将解决与蛋白质生产，回收和纯化相关的挑战。本研究的目标是开发一种高效、快速、可扩展的重组蛋白生产平台的技术和工艺。

1160184/1160117 Mayfield/NikolovScalable and low-cost production platforms are of growing importance for the development of affordable recombinant protein molecules. This is especially true in the case of complex recombinant proteins that are often difficult and expensive to produce in commercial protein production platforms, such as mammalian cell culture and E. coli. Protein production by mammalian cell cultures is still expensive because of stringent nutritional and growth requirements, specialized bioreactor design and environmental conditions, coupled with often low expression levels. E. coli, although able to produce large quantities of protein inexpensively, is generally inefficient at producing properly folded, complex proteins as soluble molecules. The single-cell eukaryotic alga, Chlamydomonas reinhardtii, has simple growth requirements (light, carbon dioxide, and salts) and its use as a production platform for complex recombinant proteins combines many of the most advantageous features of fermentation and cell cultures. The specific features of C. reinhardtii chloroplast expression include: rapid generation of stably transformed master cell lines, efficient mammalian protein folding in a non-reducing environment, a host metabolism that is tolerant of "difficult-to-express" proteins, a low propensity for undesirable post-translational modifications and formation of insoluble aggregates, cell doubling times under 10 h using completely defined protein-free inorganic media, demonstrated scalability, and absence of endotoxin and mammalian virus contamination. We have already demonstrated that C. reinhardtii is able to produce complex, multi-domain proteins that cannot be efficiently expressed in and purified from other heterologous systems. This proposal focuses on the expression and purification of two unique and therapeutically important recombinant proteins that will serve to demonstrate the utility of the platform, and that could have high-impact provided they can be produced and purified at a competitive cost. To develop C. reinhardtii as a broad and competitive platform for producing a unique class of recombinant proteins, the interdisciplinary team with complementary expertise will address challenges related to protein production, recovery, and purification. The goal of the proposed research is to develop technologies and processes for an efficient, rapid, and scalable recombinant protein production platform for using C. reinhardtii.