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

1160184/1160117 Mayfield/Nikolov可扩展和低成本的生产平台对于开发负担得起的重组蛋白分子越来越重要。 这在复杂重组蛋白的情况下尤其如此，所述复杂重组蛋白通常难以在商业蛋白生产平台（例如哺乳动物细胞培养和大肠杆菌）中生产且昂贵。杆菌 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，加上通常低的表达水平，通过哺乳动物细胞培养物生产蛋白质仍然是昂贵的。 E.大肠杆菌尽管能够廉价地生产大量蛋白质，但通常在生产适当折叠的复杂蛋白质作为可溶性分子方面效率低下。 单细胞真核生物莱茵衣原体具有简单的生长要求（光、二氧化碳和盐），并且其作为复杂重组蛋白的生产平台的用途结合了发酵和细胞培养的许多最有利的特征。 C.莱茵衣藻叶绿体表达包括：稳定转化的主细胞系的快速产生，在非还原环境中有效的哺乳动物蛋白质折叠，耐受“难以表达”蛋白质的宿主代谢，不希望的翻译后修饰和不溶性聚集体形成的低倾向，使用完全确定的无蛋白质无机培养基的细胞倍增时间低于10小时，证明了可扩展性，并且不存在内毒素和哺乳动物病毒污染。 我们已经证明了C。莱茵衣藻能够产生复杂的多结构域蛋白质，这些蛋白质不能在其它异源系统中有效表达和从其它异源系统中纯化。 该提案侧重于表达和纯化两种独特的和治疗上重要的重组蛋白，这将有助于证明该平台的实用性，并且如果它们可以以具有竞争力的成本生产和纯化，则可能具有高影响力。 发展C. reinhardtii作为一个广泛的和有竞争力的平台，用于生产一类独特的重组蛋白，具有互补专业知识的跨学科团队将解决与蛋白质生产，回收和纯化有关的挑战。 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。莱因哈德氏菌

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.