Essential Equipment for Membrane Protein Preparation and Purification

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GPCRs, also known as 7-transmembrane domain receptors, are responsible for hormonal, olfactory, visual, and neuronal signaling. Since 2007, the high-resolution X-ray crystal structures of at least a dozen distinct GPCRs have been determined, including the inactive and active states of the ß2-adrenergic receptor (ß2AR), complexed with the stimulatory GS protein. We are applying for essential apparatus so that we can maintain our facility for expression and purification of GPCRs for NMR. The key components include a bench-top centrifuge which is used extract cell pellets containing GPCRs, further purify the protein from lysate, and concentrate samples for NMR. The second component is a laminar flow hood which is used to work with cell cultures, such as insect cells which are used to express ß2-adrenergic receptor. The facility will also allow us to continue our ongoing work on 1) protein folding, 2) protein misfolding and amyloidosis, 3) enzyme functional dynamics, and 4) GPCR functional dynamics. In these studies we make use of either E coli or insect cells to express the proteins of interest. This equipment will allow us to maintain a leading role in fluorine NMR studies of complex proteins and macromolecules.