Collaborative Research: Culture of Marine Protozoans Using Industrial Bioreactors

点击翻译按钮获取中文摘要

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.