Purification of Microcystin-Binding proteins

微囊藻毒素结合蛋白的纯化

• 批准号: 538024-2019
• 负责人: Turner, Raymond
• 金额: $ 1.82万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Engage Grants Program
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=681074
• 关键词: Purification; Microcystin; Binding; proteins

Understanding water chemistry in the field remains a major challenge in a variety of industries. There exists a lack of reliable and accurate field kit technology. This makes measurements difficult for operators for water utilities, mining companies, and well owners to make timely and informed decisions about water quality. FREDsense uses bacteria and electrochemistry to create easy-to-use and portable sensors for understanding water chemistry rapidly, and directly in the field. Their first product FRED-As can detect trace levels of arsenic in the field in less than an hour. FREDsense is currently looking to expand its suite of sensors for a variety of other contaminants. Some targets such as microcystin, a potent algal neurotoxin, are overly toxic to bacteria even at trace levels and cannot be used on the current platform. Therefore, FREDsense is looking to branch into in-vitro sensors to detect a wider variety of contaminants. In this project, FREDsense will work with Dr. Turner at the University of Calgary to optimize the purification of several microcystin-binding proteins and test the function of these proteins as sensors. These proteins are large in size, and somewhat difficult to purify. Initial attempts at purification and functional testing have not been successful. Dr. Turner's research team will use their expertise in protein biochemistry to test a variety of different media conditions, host strains and growth conditions to identify optimal expression and purification conditions. Once purified, proteins will be tested for microcystin detection using standard curve assays with the proprietary FREDsense electrochemical system. If protein activity is successful, protein production infrastructure will then be transferred to FREDsense for further characterization with the goal of eventually producing a low-cost sensor for microcystin in water. This would allow end users to detect harmful algal blooms faster and more economically.

了解该领域的水化学仍然是各种行业的主要挑战。缺乏可靠和准确的现场工具包技术。这使得水务公司、采矿公司和井所有者的运营商难以及时做出有关水质的明智决策。FREDsense使用细菌和电化学来创建易于使用的便携式传感器，用于快速直接在现场了解水化学。他们的第一个产品FRED-As可以在不到一个小时的时间内检测出现场痕量的砷。FREDsense目前正在寻求扩大其用于各种其他污染物的传感器套件。一些目标，如微囊藻毒素，一种有效的藻类神经毒素，即使在痕量水平，也对细菌具有过度毒性，不能在当前平台上使用。因此，FREDsense正在寻找分支到体外传感器，以检测更广泛的污染物。在这个项目中，FREDsense将与卡尔加里大学的Turner博士合作，优化几种微囊藻毒素结合蛋白的纯化，并测试这些蛋白作为传感器的功能。这些蛋白质体积大，有些难以纯化。最初的纯化和功能测试尝试没有成功。特纳博士的研究团队将利用他们在蛋白质生物化学方面的专业知识来测试各种不同的培养基条件、宿主菌株和生长条件，以确定最佳表达和纯化条件。一旦纯化，蛋白质将使用标准曲线测定与专有的FREDsense电化学系统进行微囊藻毒素检测。如果蛋白质活性成功，蛋白质生产基础设施将转移到FREDsense进行进一步表征，目标是最终生产出水中微囊藻毒素的低成本传感器。这将使最终用户能够更快、更经济地检测有害藻华。