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Collaborative Research: Culture of Marine Protozoans Using Industrial Bioreactors

合作研究：利用工业生物反应器培养海洋原生动物

基本信息

批准号：
1701480
负责人：
Jose Fernandez-Robledo
金额：
$ 39.99万
依托单位：
Bigelow Laboratory for Ocean Sciences
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2017
资助国家：
美国
起止时间：
2017-07-01 至 2021-10-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1701480&HistoricalAwards=false
关键词：
Collaborative Research Culture Marine Protozoans

项目摘要

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.

由于这些生物体的复杂生命周期和难以获得足够量的蛋白质源材料，由细胞内寄生虫引起的疾病构成了一些独特的挑战。常规表达系统可以解决这些挑战中的一些，但以有限的方式，并且使用常规细菌、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。该项目有望为如何使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州大学进行，并纳入南缅因州社区学院的生物技术课程，鼓励学生形成和测试有关媒体成分，生长参数和纯化策略的假设。这种方法将加强和扩大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。由于这些生物体复杂的生命周期和难以获得足够数量的源材料，细胞内寄生虫蛋白质的研究带来了一些独特的挑战。传统的表达系统可以解决这些挑战中的一些，但以有限的方式。本项目旨在开发和验证一种异源蛋白表达系统，该系统不是基于传统系统，而是基于海洋原生动物Perkinsus marinus。该项目旨在将表达方案从组织培养瓶扩大到多升规模的工业相关生物反应器。将使用过程控制的生物反应器检查温度、通气、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。此外，还将检测两种生物反应器操作模式（分批和补料分批）。多种帕金虫属（Perkinsus spp.）并将菌株进行比较，以确定spp./菌株适用性/性能取决于靶基因/蛋白质。转录组学和启动子陷阱诱变将用于开发一套转染载体（例如分泌、细胞器靶向、诱导型和组成型）和工具（例如CRISPR/Cas9），其将促进感兴趣蛋白的大规模生产。该项目还旨在为P. marinus中产生的重组蛋白开发大规模纯化方案，包括评估用于亲和色谱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。它也将促进原生动物比以前更深入的表征。大蛋白质的表达是有问题的，这项技术可以提高正确表达大量这种蛋白质的能力。