A fast protein liquid chromatography (FPLC) system for purification and characterization of DMSP metabolic enzymes

用于纯化和表征 DMSP 代谢酶的快速蛋白质液相色谱 (FPLC) 系统

• 批准号: 440640-2013
• 负责人: Waller, Jeffrey
• 金额: $ 3.14万
• 依托单位: Mount Allison University
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments - Category 1 (<$150,000)
• 财政年份: 2012
• 资助国家: 加拿大
• 起止时间: 2012-01-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=506206
• 关键词: fast; protein; liquid; chromatography; FPLC

This is a reapplication for funding to buy an ÄKTA Purifier fast protein liquid chromatography (FPLC) system that is urgently needed to replace a ~20 yr old obsolete, unsupported, and inoperable FPLC. FPLCs are an essential tool for purifying and characterizing enzymes (protein catalysts). My research program seeks to discover and characterize marine algal enzymes that produce the world's most abundant organosulfur compound, dimethylsulfoniopropionate (DMSP). DMSP is vital 1) for algal cellular protection; 2) to bacteria who prefer DMSP as a sulfur source; 3) for supplying half the sulfur for the global biogeochemical sulfur cycle; and 4) to climate regulation since DMSP-derived aerosols form clouds. I exploit 3 complementary strategies (enzyme purification, comparative genomics, bacterial functional complementation assays) to discover genes encoding DMSP metabolic enzymes.The latter two approaches identify DMSP gene candidates that we will express in bacterial hosts as recombinant enzymes which then require purification. Native algal DMSP enzymes will be purified to enable protein sequencing tools which will reveal the genes encoding DMSP enzymes. These strategies all *depend* on purified, high-quality enzymes in order to confirm and study their function. Purified DMSP enzymes will be characterized with respect to their identity, biochemical structure, regulation, and kinetic behaviour. DMSP gene identification will enable phylogenetic studies of how widespread DMSP production is in marine algae and can enable studies predicting the response of algal DMSP production to future oceanic conditions. The FPLC system will see substantial use by my group (at least 1-2 postdoctoral and 15 undergraduates over my current Discovery award) and other researchers at Mt. Allison University, providing excellent HQP training in advanced protein purification technologies and supporting gene discovery efforts.

这是一个重新申请资金购买一个ÄKTA Purifier快速蛋白质液相色谱（FPLC）系统，迫切需要取代一个约20年的老过时，不支持，不能操作的FPLC。 FPLC是纯化和表征酶（蛋白质催化剂）的重要工具。 我的研究项目旨在发现和表征产生世界上最丰富的有机硫化合物，二甲基磺基丙酸酯（DMSP）的海藻酶。 DMSP是至关重要的：1）藻类细胞保护; 2）喜欢DMSP作为硫源的细菌; 3）为全球地球化学硫循环提供一半的硫; 4）由于DMSP衍生的气溶胶形成云，因此对气候调节至关重要。 我利用3种互补策略（酶纯化、比较基因组学、细菌功能互补分析）来发现编码DMSP代谢酶的基因。后两种方法鉴定DMSP基因候选者，我们将在细菌宿主中表达为重组酶，然后需要纯化。 天然藻类DMSP酶将被纯化以使蛋白质测序工具能够揭示编码DMSP酶的基因。 这些策略都“依赖”纯化的高质量酶，以确认和研究其功能。 纯化的DMSP酶将从其身份、生化结构、调节和动力学行为方面进行表征。 DMSP基因的鉴定将使系统发育研究如何广泛的DMSP生产是在海藻，并可以使研究预测藻类DMSP生产未来的海洋条件的反应。 FPLC系统将被我的团队（至少有1-2名博士后和15名本科生超过我目前的发现奖）和其他研究人员大量使用。艾利森大学，提供先进的蛋白质纯化技术和支持基因发现工作的优秀HQP培训。