CORE--MOLECULAR BIOLOGY TECHNIQUES

核心--分子生物学技术

• 批准号: 7008148
• 负责人: MARGARETHA CARRAWAY
• 金额: $ 14.18万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7008148
• 关键词: biomedical facility; biotechnology; cell line; molecular cloning; nucleic acid sequence; tissue /cell culture; transfection

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.

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。