Application for 2 shaker incubators for heterologous expression of labeled proteins

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We propose to purchase two shaker incubators for purposes of heterologous expression of labeled proteins. My lab focuses on NMR-based studies of large membrane proteins and several soluble proteins, which we make using bacterial cell cultures. Large quantities of cell culture (25 litres) are grown and later harvested to recover appropriately labeled proteins. Although some of our protein NMR samples can be grown from 1-2 litre bacterial cell cultures, we wish to express a G protein-coupled receptor (GPCR) proteins which will require 25 litres of cell culture under stringent growing conditions, to make a single NMR sample. A large fraction of our expression protocols involve cold induction and growth (4C) and we therefore request funds for 2 refrigerated shaker incubators (a 44 litre capacity). As this will be central to our work, we simply cannot move forward without an in-house shaker incubator. This apparatus would also allow us to test new ideas and strategies regarding structure/function assays of GPCRs and the determination of low resolution structures. Over 50% of all pharmacophores currently target GPCRs yet our entire protein database consists of only one high resolution structure. The importance of structural biology in this field cannot be understated and we believe that our approach can make real inroads. The equipment will also be integrated into graduate learning based laboratory modules in the summer as part of the ongoing Master of Biotechnology graduate program at the University of Toronto Mississauga and used when needed by biology and chemistry colleagues at UTM. Through this investment, we also hope to establish better contacts with local chemical and biotechnology companies with overlapping research interests as part of the Western GTA Convergence Centre, for which we will be able to uniquely contribute through the capacity to heterologously express labeled proteins on a multi-litre scale, coupled with our expertise in biosynthetic labeling, GPCR protein research, and high-field NMR.