Collaborative Research: Culture of Marine Protozoans Using Industrial Bioreactors

细胞内寄生虫引起的疾病由于这些生物复杂的生命周期和难以获得足够数量的蛋白质来源材料而带来了一些独特的挑战。传统的表达系统可以解决其中的一些挑战，但方式有限，并且使用传统的细菌，酵母或动物细胞系统特别难以产生较大的蛋白质。该项目使用一种感染牡蛎的海洋原生动物寄生虫，旨在优化这种原生动物的大量生长，并驯化它以生产疫苗验证、药物筛选和工业过程所需的大量蛋白质。该项目有望为如何利用海洋原生动物在工业相关规模上提高蛋白质产量提供新的见解，并可能产生潜在的变革性工具，用于研究和生产难以在其他系统中表达的蛋白质。该项目研究活动将在毕格罗实验室和缅因大学进行，并整合到南缅因社区学院的生物技术课程中，鼓励学生形成和测试有关介质组成、生长参数和纯化策略的假设。这种方法将加强和扩展学生的技术和智力能力，并进一步为他们继续接受教育或参与STEM劳动力做好准备。由于细胞内寄生虫复杂的生命周期和难以获得足够数量的来源材料，对细胞内寄生虫蛋白质的研究面临着一些独特的挑战。传统的表达系统可以解决其中的一些挑战，但方式有限。本项目旨在开发和验证一种基于海洋原生动物（Perkinsus marinus）而非传统系统的异源蛋白表达系统。该项目旨在扩大表达协议，从组织培养瓶到多升规模的工业相关生物反应器。温度，曝气，搅拌，盐度，pH值和营养配方对生长和蛋白质生产和回收的影响将使用过程控制的生物反应器进行检查。此外，还将测试两种生物反应器操作模式，批量和进料批量。将多个Perkinsus种和菌株进行比较，根据目标基因/蛋白确定种/菌株的适用性/性能。转录组学和启动子诱变将用于开发一套转染载体（如分泌、细胞器靶向、诱导和组成）和工具（如CRISPR/Cas9），以促进目标蛋白的大规模生产。该项目还打算开发大规模的重组蛋白纯化方案，包括评估用于亲和层析分离的各种纯化标签。这项研究可能会彻底改变口服疫苗的发展。它还将有助于比以前更深入地表征原生动物。大蛋白质的表达是有问题的，而这项技术可以提高正确表达大量这类蛋白质的能力。

Diseases caused by intracellular parasites pose some unique challenges due to the complex life cycle of these organisms and difficulty in obtaining sufficient quantities of protein source material. Conventional expression systems can address some of these challenges but in a limited way, and larger proteins are particularly difficult to produce using conventional bacterial, yeast, or animal cell systems. This project, which uses a marine protozoan parasite that infects oysters, is aimed to optimize the growth of this protozoan in large volumes and to domesticate it to produce large amounts of the proteins needed for vaccine validation, drug screening, and industrial processes. The project is expected to provide new insights on how to improve the yield of proteins at industrially-relevant scale using a marine protozoan, and could generate potentially transformative tools for use in the study and production of proteins difficult to express in other systems. The project research activity will be carried out at Bigelow Laboratory and the University of Maine, and integrated into the biotechnology curriculum at Southern Maine Community College, encouraging students in the formation and testing of hypotheses regarding media composition, growth parameters, and purification strategies. This approach will strengthen and expand the technical and intellectual abilities of the students and further prepare them for continuing their education or participation in the STEM workforce.The study of proteins from intracellular parasites poses some unique challenges due to the complex life cycle of these organisms and difficulty in obtaining sufficient quantities of source material. Conventional expression systems can address some of these challenges but in a limited way. This project seeks to develop and validate a heterologous protein expression system based not on the conventional systems, but on the marine protozoan Perkinsus marinus. The project is intended to scale up expression protocols from tissue culture flasks to multiliter scale industrially-relevant bioreactors. The effects of temperature, aeration, agitation, salinity, pH, and nutrient formulation on growth and protein production and recovery will be examined using process-controlled bioreactors. Also, two bioreactor operational modes, batch, and fed-batch will be tested. The multiple Perkinsus spp. and strains will be compared to determine spp./strain suitability/performance depending on the gene/protein targeted. Transcriptomics and promoter-trap mutagenesis will be used to develop the suite of transfection vectors (e.g. secretion, organelle targeting, inducible and constitutive) and tools (e.g. CRISPR/Cas9) that would facilitate the large-scale production of the protein of interest. The project is also intended to develop large-scale purification schemes for recombinant proteins produced in P. marinus including an assessment of various purification tags for use in affinity chromatographic separation. This research could revolutionize the development of oral vaccines. It will also facilitate much deeper characterization of protozoans than has been previously possible. The expression of large proteins is problematic, and this technology could enhance capabilities to correctly express large amounts of such protein.