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被使用的重组蛋白质。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。我们将准备用于表达未标记或标记蛋白的构建体，并纯化表达的蛋白供项目使用。在需要研究突变蛋白的地方，我们将对表达质粒进行定点诱变，然后对所得蛋白进行表达和纯化。制备过程主要涉及过氧化物还氧蛋白6的特定突变。将制备与已知多态性相匹配的谷胱甘肽s转移酶p型突变。在患者血浆中出现或增加的新发现的蛋白质将被克隆和表达，以促进抗体的产生。抗pecam抗体和Prdx6之间的融合构建体将被制备并用于表达一种靶向肺内皮的蛋白，并将测试其抗氧化应激的能力。我们还将利用Southern blotting和聚合酶链反应方法筛选小鼠尾尖DNA，以鉴定具有该基因的回交后代

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.