FPLC instrumentation for bacterial protein purification

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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.