CORE--MOLECULAR BIOLOGY TECHNIQUES

核心D设计和克隆研究人员所需的特定蛋白质的cdna的序列变化。此外，核心还建立了表达突变蛋白的细胞系。服务包括：为最佳表达系统提供建议；设计产生突变的方法；存储和维护原核和真核细胞系；维持携带野生型结构的细胞系；通过限制性内切酶和/或DNA序列分析确认野生型序列；设计用于聚合酶链式反应突变的引物；提取DNA；选择和扩增突变体；通过以下任何或所有技术确认突变：聚合酶链式反应、限制性内切酶、DNA序列和体外翻译/转录分析；转化/转染细胞系以表达突变蛋白；分离永久转染的细胞系；维护转化的细胞系。项目1要求在大肠杆菌中表达全长H-低密度脂蛋白受体和8个末端不同结构域的截短片段，用于生物物理研究，包括CD光谱、量热和低温电子显微镜。项目2要求哺乳动物细胞系中的19个载脂蛋白变异体研究脂蛋白的组装和MTP在这一过程中的作用。为了用核磁共振和圆二色谱进行结构分析，截短的apoB-5.9将在杆状病毒中表达。项目3用12个新结构分析了apoA-I的结构、稳定性和构象适应性，包括：apoA-I的缺失、稳定点突变、3个44个残基和2个共同的多肽片段。项目4正在研究四种脂肪酸结合蛋白的动力学和配体结合：ILBP、肝脏FABP和人类肠道FABP的两个等位基因。通过在大肠杆菌中表达，将产生用于这些研究的未标记蛋白质。CORE D的所有人员将对新用户进行细胞培养技术培训，并监督细胞培养设施的正确使用。这将确保电池系统的安全维护，并保证遵守NIH的指导方针。

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.