Core D designs and clones sequence changes in the cDNA of specific proteins required by the investigators. In addition, the core establishes cell lines expressing the mutated proteins. The services includes: advice toward an optimal expression system; design methodologies to create mutations; store and maintain prokaryotic and eukaryotic cell lines; maintain cell lines carrying wild type constructs; confirm wild type sequence by restriction and/or DNA sequence analysis; design primers for PCR mutagenesis; extract DNA; select and amplify mutants; confirm mutations by any or all of the following techniques: PCR, restriction digests, DNA sequence and in vitro translation/transcription analysis; transform/transfect cell lines to express the mutated protein; isolate permanently transfected cell lines; maintain transformed cell lines. Project 1 has requested full-length H-LDL-receptor and eight truncations terminating in different domains to be expressed in C. coli for biophysical studies including CD spectroscopy, calorimetry and cryoelectron microscopy. Project 2 requests nineteen apoB variants in mammalian cell lines to study the assembly of lipoproteins and the role of MTP in the process. For structural analysis with NMR and CD spectroscopy a truncated apoB-5.9 will be expressed in Baculovirus. Project 3 analyzes the struture, stability and conformational adaptability of apoA-I with twelve new constructs including: deletion, stabilizing point mutations, three 44-residue and two consensus peptide fragments of apoA-I. Project 4 is studying dynamics and ligand binding of four fatty acid binding proteins: ILBP, liver-FABP and two alleles of human intestinal FABP. Unlabeled proteins will be generated for these studies by expression in E. coli. All personnel in Core D will train new users in cell culture techniques and oversee proper use of cell culture facilities. This will ensure safe maintenance of cell systems and guarantee compliance with NIH guidance.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Program Projects (P01)
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Heart, Lung, and Blood Initial Review Group (HLBP)
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Boston University
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Mei, Xiaohu; Atkinson, David (2015) Lipid-free Apolipoprotein A-I Structure: Insights into HDL Formation and Atherosclerosis Development. Arch Med Res 46:351-60
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