CORE--Molecular Engineering and Protein Preparation

核心--分子工程和蛋白质制备

• 批准号: 7796694
• 负责人: SHELDON I FEINSTEIN
• 金额: $ 35.67万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7796694
• 关键词: Adenoviruses; Alleles; Animal Husbandry; Antibodies; Antibody Formation; Antioxidants; Area; Backcrossings; Breeding; Cell Culture Techniques; Cells; DNA; Endothelium; Engineering; Escherichia coli; Genes; Genetic Polymorphism; Glutathione S-Transferase; Goals; In Vitro; Knock-out; Lung; Methods; Molecular; Molecular Biology; Mus; Mutate; Mutation; Oxidative Stress; Patients; Plasma; Plasmids; Polymerase Chain Reaction; Preparation; Protein Engineering; Proteins; Reactive Oxygen Species; Recombinant DNA; Recombinant Proteins; Recombinants; Site-Directed Mutagenesis; Southern Blotting; Tail; Testing; Virus; interest; mutant; offspring; peroxiredoxin; protein expression

The primary goal of the Molecular Engineering and Protein Preparation, is to produce and test recombinant proteins that will be used. In most cases, this will involve engineering plasmids for expression of the proteins of interest or mutating genes and expressing them, then inducing protein expression, purifying the protein obtained and activating it or testing it for activity. We will prepare constructs for expressing untagged or tagged proteins and purify the expressed proteins for use by the projects. Where there is a need to study mutant proteins, we will perform site-directed mutagenesis of expression plasmids followed by expression and purification of the resulting proteins. Preparations will primarily involve specific mutations in peroxiredoxin 6. Mutations in the p form of glutathione S-transferase that match known polymorphisms will be prepared. Newly identified proteins that appear or increase in plasma from patients will be cloned and expressed in order to facilitate antibody production. A fusion construct between anti-PECAM antibody and Prdx6 will be prepared and used to express a protein that will target the lung endothelium and will be tested for its ability to protect against oxidative stress. We will also utilize Southern blotting and polymerase chain reaction methods to screen mouse tail tip DNA in order to identify offspring from backcrosses that have the knockout allele or double knockouts from breeding of two independent knockout lines. There may be a need for adenoviral constructs in order to introduce an expression construct into a high percentage of cells in primary cultures. We will prepare recombinant adenovirus using a recently developed recombinant DNA method which allows in vitro construction and DNA preparation in E. coli. The DNA will then be packaged into virus and amplified using HEK 293 cells supplied by the Cell Culture and Animal Husbandry. Finally, we will supply molecular biology expertise in areas of need.

分子工程和蛋白质制备的主要目标是生产和测试将使用的重组蛋白。在大多数情况下，这将涉及工程质粒用于表达感兴趣的蛋白质或突变基因并表达它们，然后诱导蛋白质表达，纯化获得的蛋白质并激活它或测试它的活性。我们将准备表达未标记或标记蛋白质的构建体，并纯化表达的蛋白质供项目使用。如果需要研究突变蛋白，我们将对表达质粒进行定点诱变，然后表达和纯化所得蛋白。制备将主要涉及过氧化物氧还蛋白6中的特定突变。将制备与已知多态性匹配的谷胱甘肽S-转移酶p型突变。将克隆和表达在患者血浆中出现或增加的新鉴定的蛋白质，以促进抗体产生。将制备抗PECAM抗体和Prdx 6之间的融合构建体，并用于表达靶向肺内皮的蛋白质，并测试其保护免受氧化应激的能力。我们还将利用Southern印迹和聚合酶链反应方法来筛选小鼠尾尖DNA，以鉴定来自回交的后代， 敲除等位基因或来自两个独立敲除系育种的双敲除。可能需要腺病毒构建体以将表达构建体引入原代培养物中的高百分比细胞中。我们将使用最近发展的重组DNA方法制备重组腺病毒，该方法允许在体外构建和在E.杆菌然后将DNA包装到病毒中，并使用由细胞培养和动物饲养部提供的HEK 293细胞进行扩增。最后，我们将在需要的领域提供分子生物学专业知识。