Things are moving fast in the lab which is exciting as it means the project is moving forward quickly too. However, it does mean that I now have lots of data to share in this post!
Firstly, thanks to very generous sharing of reagents from Dr. Ihn Sik Seong, we are now able to make the complete full-length huntingtin protein in the lab with 3 different polyQ lengths using an insect cell expression system. Ihn Sik has worked on huntingtin structure-function for many years now, you can read more about his great science on his page. Thanks again Ihn Sik!
We were able to replicate Ihn Sik’s expression and purification of the full-length huntingtin, first in a small-scale trial experiment. Next I worked to try and optimise the purification protocol to make a quality protein sample which we can then use for analysis and further experiments. The first few attempts did not work very well – the protein was very aggregated meaning that all of the protein molecules were sticking to each other which is not ideal for subsequent experiments. However, trying a few different conditions to see which might help purify the huntingtin protein more stringently, I landed on a set of conditions which work really well and make a sample which looks similar to that which Ihn Sik describes in his paper. Since then I have optimised a procedure called gradient fixation (Grafix) which chemically fixes the huntingtin protein to make it more stable, possibly a critical step in generating a sample for structural analyses.
Huntingtin protein fractions from the grafix procedure were confirmed to be stable monomeric huntingtin by analysis on a gel filtration column.
I have sent purified huntingtin protein sample, untreated and Grafix treated, to Professor Susan Lea, University of Oxford. Susan specialises in structural biology of various interesting proteins, using the technique most suitable to the sample. In the case of huntingtin, this is cryo electron microscopy (cryo-EM) as the protein is very big. Susan is in the process of setting up the huntingtin samples on special grids which she can look using an electron microscope, this should definitively tell us whether the sample is any good or not. I am keeping my fingers crossed!