Description of the Protein Analytical Core
The Protein Analytical Core provides facilitiesfor the purification and characterization of proteins including staff available to assist investigators in these tasks. These include a fluorometer, dynamic light scattering instrument, spectropolarimeter, analytical ultracentrifuge, chromatographic apparatus, isothermal titration calorimeter, stopped flow kinetic apparatus, and X-ray crystallography instrumentation. The core also maintains equipment for high-level production and purification of recombinant proteins from bacteria, yeast, and insect cells. The core provides training as requested, but allows groups with prior training to use equipment as needed.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 are useful for assessing the degree of correct folding of recombinant proteins. The light scattering instrument as well as a fluorometer (PerkinElmer LS55) are available in the core and can be used to monitor macromolecular interactions. The fluorometer can also be used to monitor conformational changes and assay enzyme activity. An isothermal titration calorimeter (MicroCal VP-ITC) is 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.
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 available 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 BBSRB 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.
Protein Expression and Purification. 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 cell paste. Large capacity preparative centrifuges and rotors as well as benchtop centrifuges are also maintained in the core facility. Protein purification equipment includes several computer controlled AktaExplorer (GE Healthcare) automated chromatography systems and associated high capacity chromatography columns. These systems are appropriate for both small- and large-scale protein purification. Equipment for protein purification is located on the second floor of BBSRB.