CORE--PROTEIN EXPRESSION AND CRYSTALLOGRAPHY

University Of California San Diego, La Jolla CA

Investigators

Linked publications & trials

Abstract

DESCRIPTION (provided by applicant): Two years ago, we realized that a rate-limiting step for many of our Program Project Grant projects was lack of large amounts of pure and soluble proteins. This is necessary for all of the structural studies that are being proposed. Therefore, we have added a "protein expression" component to the "crystallography" core and changed its name to "Protein Expression and Crystallography Core." Using funds and equipment from other sources, we have installed in Urey Hall adjacent to the crystallography laboratory, a Protein Expression laboratory. This laboratory is now fully functional and is able to produce routinely large amounts of pure and soluble proteins or protein domains for the Taylor, Jennings and Newton laboratories. In addition to the routine production of many proteins needed for structural studies, the Protein Expression is recloning a number of proteins and introducing them into more easily cleavable vectors. The Protein Expression Laboratory has also worked closely with the Mass Spectrometry Core facility in Urey Hall to identify stable domains that will be good candidates for crystallization and NMR. While initially we focused on smaller domains, we are now expressing larger proteins. Another goal for the Expression Core lab is to establish and optimize expression and purification conditions for 15N/13C labeled proteins. For these preps, we shall be using fermenters to optimize our yields. The Crystallography Core is also very well equipped. We have a state-of-the-art facility that is well staffed with scientists whose expertise is fully available to the investigators of this Program Project. Dr. Madhusaden will continue as project leader and will serve as an interface between each project and the Core. A continuing goal of the Crystallography Core is to crystallize and solve the structures of the AKAP Binding Domains of RIalpha, RIIalpha, and RIIbeta with the PKA Binding Domains of the Dual Specific A Kinase Anchoring Proteins (DAKAP-1 and DAKAP-2) (Taylor and Jennings). We are making excellent progress with the complex of the Dimerization/ Docking domain of RIalpha and RIIalpha with RPP8 and RPP7. A long-term goal is to crystallize the PKA Binding Domains from DAKAP-1 (RPP7) and DAKAP-2 (RPP8) with full-length R subunit or holoenzymes. Other major projects during the next granting period are to crystallize and solve the structure of the N-terminal segment of HSP70, and the PKC core (Newton ), and PKC Binding Domain of Pericentrin (Newton ).