CORE--MOLECULAR BIOLOGY TECHNIQUES

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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.