CORE--Molecular Engineering and Protein Preparation

分子工程和蛋白质制备的主要目标是生产和测试将要使用的重组蛋白质。在大多数情况下，这将包括设计表达感兴趣的蛋白质或突变基因的质粒并表达它们，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的蛋白质的构建体，并纯化表达的蛋白质以供项目使用。在需要研究突变蛋白的地方，我们将对表达载体进行定点突变，然后表达和纯化所产生的蛋白。准备工作将主要涉及过氧化还蛋白6的特定突变。将准备与已知多态相匹配的谷胱甘肽S转移酶p型突变。新发现的患者血浆中出现或增加的蛋白质将被克隆和表达，以促进抗体的产生。将制备抗PECAM抗体和Prdx6之间的融合构建物，并用于表达一种针对肺内皮细胞的蛋白质，并将测试其抵御氧化应激的能力。我们还将利用Southern杂交和聚合酶链式反应的方法来筛选小鼠的尾尖DNA，以便从具有 来自两个独立的基因敲除系育种的基因敲除等位基因或双基因敲除。可能需要腺病毒构建体以将表达构建体引入原代培养的高百分比细胞中。我们将使用最近发展的一种重组DNA方法来制备重组腺病毒，这种方法可以在体外构建并在大肠杆菌中制备DNA。然后，DNA将被包装成病毒，并使用细胞培养和畜牧业提供的HEK 293细胞进行扩增。最后，我们将在有需要的领域提供分子生物学专业知识。

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.