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Protein Analytical Core Administrative | Protein Analytical | Imaging | Organic | Proteomics | Viral Production |
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DESCRIPTION OF THE PROTEIN ANALYTICAL CORE Introduction The projects of the COBRE investigators involve characterization of recombinantly produced proteins. The Protein Analytical Core provides well-maintained facilities, as well as a helping hand, to simplify these tasks and provide enhanced capabilities. In particular, the core provides protein characterization as well as protein production and purification equipment not normally available to junior investigators. Our approach with regard to the COBRE investigators is to maintain the major equipment and help to train new users but allow the members of the investigators' groups to use the equipment directly with priority access. In this way, we keep personnel and supplies costs for the core to a minimum while still giving the COBRE investigators a significant advantage in their research. Our specific objectives for this core are to:
Current Facilities Protein Characterization Subcore Protein analytical instrumentation. A number of instruments for protein characterization are maintained in the core. A Jasco J-810 spectropolarimeter is available for circular dichroism measurements to characterize secondary structure content. A dynamic light scattering instrument (Protein Solutions DynaPro 99) can be used to assess molecular weight and degree of monodispersity. Both of these techniques will be useful for assessing the degree of correct folding of recombinant proteins. The light scattering instrument as well as the fluorometer (PerkinElmer LS55) available in the core can be used to monitor macromolecular interactions, and the fluorometer can also be used to monitor conformational changes and assay enzyme activity. An isothermal titration calorimeter (MicroCal VP-ITC) is also available for characterizing binding interactions. These capabilities are complemented by an analytical ultracentrifuge (Beckman Coulter XL-A). Transient state kinetics can be investigated with the use of the core's Kintek SF-2001 pneumatically driven stopped-flow instrument. A Waters Breeze HPLC is available for characterization of small molecules, peptides, and proteins. Most of the analytical instrumentation is located in one room of the BBSRB building that houses the Department of Molecular and Cellular Biochemistry. Crystallography. X-ray crystallography instrumentation includes a Rigaku RU-200 X-ray generator with two R-AXIS IV++ image plate detectors and graded multilayer optics. Two X-STREAM cryosystems are provided for sample cooling. A TTP Labtech Mosquito crystallization robot is used to set screens for crystallization conditions. Associated incubators maintained at various temperatures and a dedicated cold room are used for incubating crystal trays. A membership in the SER-CAT sector at the Advanced Photon Source (Argonne National Laboratory) is administered through the core, providing access to state-of-the-art X-ray data collection facilities. A total of 288 hours/year of beam time at SER-CAT is available for researchers on campus. The local X-ray facility is located in the basement of the building. Computational capabilities. A number of computer workstations and a 16 node dual Xeon processor Linux cluster (Microway) are available for computational support. Dedicated computer workstations are located in B272 and the Linux cluster is on the first floor in B131. In addition, the core has access to the University of Kentucky's High Performance Computing Complex, which includes an IBM/X-series cluster with 340 nodes and 680 dual core processors that is integrated with a group of large shared memory processor machines and a large mass storage system. Software for crystallography, NMR analysis, computational chemistry, and related approaches is available through our membership in the Harvard SBGrid initiative, which also provides access to additional computer clusters. Proetin Expression and Purification Subcore Expression of recombinant proteins. The core maintains two New Brunswick BioFlo 3000 ten liter fermenters with associated gas and exhaust systems. Each fermenter has a dedicated recirculating cooling/heating system that allows culture at a range of temperatures. Additional vessels are available so that these instruments can be used for bacterial, yeast, and mammalian cell culture. The core is equipped with two laminar flow hoods outfitted with appropriate filters and UV sterilization lights for culture work. Large and small incubators are available for insect and mammalian cell culture. Core instrumentation for protein production and cell culture is largely located in nearby rooms on the second floor of the building that houses the Department of Molecular and Cellular Biology. Protein purification. A French press and sonicator are available for disrupting small volumes of cell paste, and a continuous flow microfluidizer (Microfluidics Corporation model 110) is available for or disrupting larger quantities of paste. Large capacity preparative centrifuges and rotors as well as benchtop centrifuges are also maintained in the core facility. Protein purification equipment includes a computer controlled AktaExplorer (GE Healthcare) automated chromatography system and associated high capacity chromatography columns. This system is appropriate for both small- and large-scale protein purification. Equipment for protein purification is located in rooms B216C and B. Personnel and Management The Core will be directed by Dr. David Rodgers (5% effort), an experienced enzymologist and x-ray crystallographer. He will oversee the daily operation of the core and will consult with the COBRE investigators and their research teams on the use of the core facilities. Dr. Jack Schmidt (75% effort), who is experienced in analytical techniques as well as protein expression and purification, will maintain the core facilities and assist users when necessary. Dr. Schmidt will oversee scheduling and provide hands-on training in the use of equipment. His role in maintaining the equipment and training new users is particularly critical in this multi-user facility. Core Use by COBRE Projects Project 1: Structural Studies of Neuropilin Signaling. Craig W. Vander Kooi, PI Dr. Vander Kooi will use the Protein Analytical Core extensively in his research project. He will be producing VEGF and semaphorin ligand and receptor constructs using the protein production and purification equipment. He will also be using the analytical instrumentation to characterize the interactions between the receptors and their ligands as well as the higher order complexes formed. He will make extensive use of the crystallization and X-ray crystallography facilities, since X-ray structure determination of various complexes is a major goal of his project. Project 2: Deciphering the connection between Lafora disease and AMP-activated protein kinase/Wolff-Parkinson-White syndrome. Matthew S. Gentry, PI Dr. Gentry will use the Protein Core in producing various recombinant constructs of laforin and AMP kinase. He will also use the analytical instrumentation in studies of the regulation of laforin by phosphorylation. Dr. Gentry will also pursue crystal structure determination with some of his constructs using the core facilities. Project 3: Negative Regulation of the Yeast MRP, Ycf1p, and Human MRPs by CKII. Christian M. Paumi, PI In his studies of the multidrug resistance-associated protein (MRP), Dr. Paumi will make use of the Protein Core in producing and purifying recombinant wild type and mutant Ycf1p, the yeast MRP, as well as the Cka1p kinase subunit. In addition, he will make use of the analytical instrumentation to characterize the kinetics of phosphorylation of Ycf1p in vitro. Project 4: Mechanisms of Peripheral Regulatory T Cell Generation: Role of Polyunsaturated Fatty Acids. Francesc Marti, PI Dr. Marti will make use of cell culture facilities in the core for the growth and maintenance of T cell populations. Impact of the Core on the Broader Research Community The Protein Analytical Core has served 25 research groups from the University of Kentucky community and the surrounding area, including nine groups associated with the COBRE proposal. The core maintains a web site that serves to inform the community of the instrumentation and services available.
Principal Investigators and Affiliations of groups using the Protein Analytical Core
Future Plans
We anticipate that the Protein Analytical Core will continue to grow and develop to better serve the COBRE
faculty and the local research community. We will work to expand the range of biophysical instrumentation and
techniques available in the core. Recently, we have submitted an NSF proposal that if funded would add
lifetime fluorescence, differential scanning calorimetry, and surface plasmon resonance instrumentation to the
core. In addition, the proposal would increase our crystallography automation with the addition of additional
robotics and visualization equipment for high throughput crystallization trials. We are currently installing
capabilities for remote X-ray data collection in conjunction with the SER-CAT facility at the Advanced Photon
Source. The ability to collect data remotely will allow us to make more efficient and frequent use of the
exceptional data collection facilities at SER-CAT. The Protein Analytical Core has proven to be an important
resource for the University of Kentucky research community. As part of the Center for Structural Biology, it is
an official university center, and we anticipate long term financial support for the core from a number of
mechanisms, including user fees, center grants, program project grants, and institutional shared resource
support.
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