Engineering a plant blue copper protein from human Ephrin A1
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The goal of this project is to prove an evolutionary relatedness between two protein families; metazoan Eprhins and plant blue copper binding proteins known as phytocyanins. Since members these two protein families share remarkably similar folding topologies, we suspect such relationship exists. Our plan is to express a quadruple mutant human Ephrin A1 protein and a blue copper binding Early Nodulin-13 (EN-13) plant protein. Once isolated, we planed to test copper binding capabilities of these two proteins. If our engineered Ephrin protein was able to bind copper it would suggest an evolutionary relatedness between them and phytocyanins which are blue copper binding proteins. First, E. coli bacteria were transformed with vectors containing the gene encoding for each of the proteins of interest. We used the pET-expression system to produce large quantities of our proteins. We were then able to express both the quadruple mutant human Ephrin A1 protein and the wild type EN-13 plant protein. Isolation of the proteins revealed that they had been stored in inclusion bodies by the E. coli cells. In order to obtain an active form of the proteins, we attempted to refold the proteins. We were unable to successfully refold the proteins, therefore, we were unable to test the copper binding capabilities of the quadruple mutant human Ephrin A1. We suspect that the refolding problem may have been due to the fact that we had used a prokaryotic system for expression of the proteins. We will consider expressing these proteins in an insect system. This will ensure that eukaryotic post-translational modifications will be made such as glycosylation.