FPLC instrumentation for bacterial protein purification

我们的研究小组研究了抗菌剂化合物的抗药性，包括防腐剂、DNA螯合染料和重金属。通过这些问题，我们探索了在细胞膜中发现的调节这些抗性的蛋白质。我们还研究了蛋白质如何跨膜移动，以及电子转移蛋白质--细胞中的能量机械--如何聚集在一起并成熟为一种功能形式。为了研究这些系统，我们使用了色谱学工具，这是生命科学实验室中的一项关键技术，用于纯化、评估、鉴定和表征分子和细胞。我们小组使用了各种层析方法，包括但不包括：离子交换法、反相法、尺寸排除法、亲和力和伪亲和力。这些方法中的每一种都根据分子的化学性质来分离分子。目前的赠款要求提供资金购买快速蛋白质液相色谱(FPLC)系统。这样的系统使我们能够提纯蛋白质、多肽和氨基酸，以及蛋白质的交换缓冲区。完整的膜蛋白存在于细胞膜中，很难处理，因为它们需要洗涤剂来保持它们的溶解。它们也有少量存在。因此，需要付出相当大的努力才能提纯到足以表征其结构和功能的水平。当我们研究参与电子转移蛋白成熟的蛋白质时，我们进行突变实验，改变氨基酸以了解蛋白质-蛋白质的相互作用。因此，每个突变蛋白必须与亲本蛋白一起提纯，以便在各种生物物理和生化实验中进行比较。在这些项目之间，需要我的团队提纯大量的蛋白质。FPLC是实现这一点的最好和最有效的方式。超过15名研究人员和研究实习生将不断使用所要求的FPLC。

Our research group studies resistance to antimicrobial compounds including antiseptics, DNA inter-chelating dyes and heavy metals. Through these questions we explore proteins found in the cell membrane that mediate these resistances. We also study how proteins move across membranes and how electron-transfer proteins - the energy machinery in the cell - come together and mature into a functional form. In order to study these systems we employ the tool of chromatography, which is a key technique in life science laboratories and is used to purify, evaluate, identify, and characterize molecules and cells. Our group uses a variety of chromatography methods including but not inclusively: ion exchange, reverse phase, size-exclusion, affinity and pseudo-affinity. Each of these methods separate molecules based on their chemical properties. The present grant asks for funds for the purchase of a Fast Protein Liquid chromatography (FPLC) system. Such a system allows us to purify proteins, peptides and amino acids as well as exchange buffers of the proteins. Integral membrane proteins are found in cell membranes and are very difficult to work with, as they require detergents to keep them soluble. They are also found in small quantities. Therefore, considerable effort is required to purify amounts at levels high enough to characterize their structures and functions. As we study proteins involved in the maturation of electron transfer proteins we perform mutagenesis experiments changing amino acids to understand the protein-protein interactions. Thus, each mutant protein must be purified along with the parent protein form to compare in various biophysical and biochemical experiments. Between these projects, a large number of proteins need to be purified by my group. An FPLC is the best and most efficient manner to do this. Over 15 researchers and research trainees will find constant use for the FPLC requested.