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.
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