The main role of the Protein Core will be to produce proteins for each of the Bridge and Pilot Projects. Furthermore, the Core will be accessible to investigators outside the consortium who are seeking help to develop methods for high yield expression of specific membrane proteins, detergent solubilization optimization, and purification of protein targets. The emphasis of the Core will be preparation of homogeneous functional proteins for use in studies of structure, dynamics and mechanism. Core D1 members will cooperate in using their collective expertise to develop and optimize new expression protocols for proteins used by the Consortium. The Core will utilize its expertise and technologies already in place to provide a wide range of expression systems, both prokaryotic and eukaryotic expression in cells or cell-free, to accommodate the specific requirements of each protein target. Because none of the proposed studies can be accomplished without protein, we will use our technologies from the onset of this project to provide samples for the Consortium as soon as possible. In general, membrane proteins present challenges at every step of the way to structural determination. The synthesis and processing of these proteins is complex and often involves specific folding factors or chaperones [1, 2]. Expression in bacteria is favored because of the low cost to grow large culture volumes by fermentation, the potential for high yields, the very fast growth rate, and the simplicity and flexibility of expression systems. However, intrinsic differences in how proteins are processed often prevent the expression of adequate amounts of protein with the proper fold, and it may not be possible to isolate and purify sufficient quantities in a homogenous state. For these reasons, the Protein Core will employ a range of expression systems including preparative scale cell-free systems, which should provide a high probability that any selected protein target can be produced. In addition, we describe high throughput screening methods to further enhance and optimize expression. Because the Consortium emphasizes the study of proteins for which a high resolution structure is already accomplished, such proteins have already been successfully expressed at high levels and homogeneous purified protein prepared. We will take advantage of the information already available, but in some cases targets are proposed for study, in particular mutant forms, that have not been expressed at high levels. Furthermore, investigators may require the protein target in a specific environment or state. For example, the protein may be part of a complex, which requires expression of multiple proteins, some of which do not express in bacteria. This example may require simultaneous expression of all the components in a mammalian or insect cell for proper processing. The Protein Core will apply a range of expression strategies taking into consideration several needs: the quantity, the multimeric state, the level of purification, and the modifications required for manipulations. Of foremost importance is the functionality of the protein. For this reason, the Protein Core will work closely with all investigators to assure that the activity of each target is properly assessed.
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| Sun, Chang; Benlekbir, Samir; Venkatakrishnan, Padmaja et al. (2018) Structure of the alternative complex III in a supercomplex with cytochrome oxidase. Nature 557:123-126 |
| Mahinthichaichan, Paween; Gennis, Robert B; Tajkhorshid, Emad (2018) Cytochrome aa3 Oxygen Reductase Utilizes the Tunnel Observed in the Crystal Structures To Deliver O2 for Catalysis. Biochemistry 57:2150-2161 |
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