Collaborative Research: Development of an Integrated Eukaryotic Algae Platform for Difficult-to-produce Therapeutic Molecules

1160184/1160117梅菲尔德/尼科洛夫可伸缩和低成本的生产平台对于开发负担得起的重组蛋白质分子越来越重要。对于在哺乳动物细胞培养和大肠杆菌等商业蛋白质生产平台上生产往往困难和昂贵的复杂重组蛋白来说，情况尤其如此。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，再加上通常低表达水平，哺乳动物细胞培养物生产蛋白质的成本仍然很高。大肠杆菌虽然能够以低廉的价格生产大量蛋白质，但在将适当折叠的复杂蛋白质作为可溶性分子生产方面通常效率低下。单细胞真核藻，衣藻，具有简单的生长要求(光、二氧化碳和盐)，作为生产复杂重组蛋白的平台，结合了发酵和细胞培养的许多最有利的特征。莱茵梭菌叶绿体表达的具体特征包括：快速产生稳定转化的主细胞系，在非还原环境中高效地折叠哺乳动物蛋白质，宿主代谢对难以表达的蛋白质具有耐受性，对翻译后修饰和形成不可溶聚集体的倾向低，使用完全定义的无蛋白质无机培养基细胞倍增时间在10h以下，表现出可扩展性，以及没有内毒素和哺乳动物病毒污染。研究人员已经证明，莱茵梭菌能够产生复杂的、多结构域的蛋白质，而这些蛋白质无法在其他异源系统中高效表达和纯化。这项提议的重点是表达和纯化两种独特的、具有治疗重要性的重组蛋白，这两种重组蛋白将用于展示该平台的实用性，并且如果它们能够以具有竞争力的成本生产和纯化，则可能产生高影响。为了将莱茵哈迪氏杆菌发展成为生产一类独特重组蛋白质的广泛和具有竞争力的平台，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。拟议研究的目标是开发高效、快速和可扩展的重组蛋白生产平台的技术和工艺，以利用莱茵哈迪尔酵母。

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. The investigators 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.