GFP Production and Purification

Huntington’s Disease (HD) is a hereditary neurodegenerative disease. The cause of this disease is a CAG repeat extension in the HTT Gene. This extension is then translated into an elongation of exon 1 which is primarily composed of a disordered PolyQ repeat. Although the cause is known, the mechanism by which this extension affects the function of Huntingtin (HTT), the protein produced by the HTT Gene, has yet to be understood. A part of this difficulty is our lack of understanding of the role of normal HTT in our cells. During the production of pure HTT sample, we began to notice an accumulation of nucleic acid material. To test the identity of this material, we sent it to one of our collaborators who told us it was RNA. Now, we are interested in preforming CLIP-seq to identify the specific sequence bound to HTT. Standard expression methods have been found to have minimal success due to the sheer size of HTT so other methods are being explored. One of which, is inserting formed protein into the neuronal cells via electroporation as described by Lambert, H. et. al. in Electroporation - mediated uptake of proteins into mammalian cells . By importing tagged protein into neuronal cells, we hope to capture a more readable RNA sequence, giving us a clue about its cellular function. To get the protein into the cell, we are planning to use electroporation. This process can be quite variable dependent upon the equipment used and the cells used, etc. we are using GFP to optimize conditions as it is quite easy to image in cells. In order to continue with this, a concentrated GFP sample must be made.