A fermentation system is requested, comprising a pilot scale fermentor for cell growth and a continuous flow centrifuge for cell harvest. This system will replace existing equipment that is 30 years old and lacks many capabilities of modern fermentors. There is no other such fermentation system available at Stanford University. It will be used for the production of proteins for biochemical and structural studies, primarily in four research areas, gene expression, cell signaling, and the immune response. Studies of gene expression are performed by the Kornberg laboratory on transcription and by the Puglisi laboratory on protein synthesis. Kornberg and colleagues investigate the structure and regulation of the RNA polymerase II transcription machinery from the yeast Saccharomyces cerevisiae. This research has been critically dependent for several decades on large scale fermentation, to obtain the quantities of proteins needed for biochemistry and crystallography. It is now proposed to bring the entire effort to fruition, and a modern fermentor is needed for the purpose. The Puglisi group study mechanisms of protein synthesis in prokaryotes by NMR and fluorescence methods. Large amounts of ribosomes, accessory proteins, and tRNAs are required. Fermentation on the scale requested will greatly facilitate the work. Weis and colleagues study cell-cell interaction, cell signaling, and membrane transactions. They are currently pursuing the molecular basis of cell adhesion through the surface receptor, and binding of regulatory proteins to the SNAREs responsible for cell membrane fusion. All of this work entails X-rays crystallographic analysis, and depends on bacterial fermentation. The studies of Wnt interaction particularly require low temperature cell growth. The Jardetzky group study viral entry glycoproteins, IgE antibodies and receptors, and TGF-B ligands and receptors. Many of the proteins are expressed in bacteria, and will benefit from large scale cell growth for eventual structural studies by X-ray crystallography.
The proposed fermentation system will enable research directed towards the mechanisms of gene expression, cell signaling, and the immune response. Growth of cell cultures on a large scale in the proposed system will permit the isolation of proteins involved in these fundamental processes. The isolated proteins will be subjected to biochemical and structural analyses.
| Oberthuer, Dominik; Knoška, Juraj; Wiedorn, Max O et al. (2017) Double-flow focused liquid injector for efficient serial femtosecond crystallography. Sci Rep 7:44628 |
| Robinson, Philip J; Trnka, Michael J; Bushnell, David A et al. (2016) Structure of a Complete Mediator-RNA Polymerase II Pre-Initiation Complex. Cell 166:1411-1422.e16 |
| Robinson, Philip J; Trnka, Michael J; Pellarin, Riccardo et al. (2015) Molecular architecture of the yeast Mediator complex. Elife 4: |
| Murakami, Kenji; Tsai, Kuang-Lei; Kalisman, Nir et al. (2015) Structure of an RNA polymerase II preinitiation complex. Proc Natl Acad Sci U S A 112:13543-8 |
| Liu, Xin; Bushnell, David A; Kornberg, Roger D (2013) RNA polymerase II transcription: structure and mechanism. Biochim Biophys Acta 1829:2-8 |
| Murakami, Kenji; Elmlund, Hans; Kalisman, Nir et al. (2013) Architecture of an RNA polymerase II transcription pre-initiation complex. Science 342:1238724 |
| Robinson, Philip J J; Bushnell, David A; Trnka, Michael J et al. (2012) Structure of the mediator head module bound to the carboxy-terminal domain of RNA polymerase II. Proc Natl Acad Sci U S A 109:17931-5 |
