Scientific Core 1:Human Clinical and Protein Engineering Core

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Scientific Core 1(SC1). Project Summary/Abstract Each of the three Projects within the Program Project will require purified native and oxidatively modified lipoproteins, access to human clinical material with known clinical follow-up and a source of native, mutant or site-specific post-translationally modified proteins of interest to use in their studies. The Human Clinical Material & Protein Engineering Core (Scientific Core 1; SC1) will provide all of these services, allowing for expansion of the scope, scale and clinical significance of proposed studies in each Project of this Program. The Human Clinical Material & Protein Engineering Core has 3 primary objectives: First, the core will facilitate human clinical and translational studies of Program investigators by providing the following services: (i) Coordination of all IRB paperwork, advertising / consenting of volunteers, contact of patients with specific phenotypes (e.g. high LP(a) levels), and phlebotomy services. (ii) Using standardized SOP for isolation and characterization of the following human biological materials: whole blood (e.g. for PMN and MPO isolation); plasma; serum; isolated lipoproteins (e.g. VLDL, LDL, HDL and LP(a)). (iii) Coordination of monocyte isolation via elutriation. (iv) Coordination of access to archival human clinical specimens (plasma, serum, DNA) from the GeneBank cohort for proposed clinical studies. (v) Access to human clinical specimens available within the Center for Cardiovascular Diagnostics & Prevention. Second, the core will assist Program investigators with protein engineering and antibody generation by providing the following services: (i) assist investigators with design, expression, and purification of distinct classes of wild type and mutant forms of proteins. (ii) Generation of low endotoxin recombinant proteins for in vivo studies. (iii) Orthogonal amino acid incorporation using tRNA mutants to specifically form individual recombinant proteins harboring unnatural amino acids (e.g. nitrotyrosine, chlorotyrosine, Nε-acetyl-lysine). And (iv) assistance in generation of both polyclonal and monoclonal antibodies. Finally, the core will serve as an educational and training hub for Program investigators, especially students and fellows. Centralizing these aspects of the work and training will greatly enhance the significance, innovation, and clinical relevance of studies performed in each Project of this Program.