I am back from vacation and other distractions and now have some new and exciting data to share with everyone.
Following on the previous limited proteolysis experiment where I chopped up the protein into proteolyticaly stable nuggets which allowed me to work out the potential domain boundaries of the huntingtin protein, I designed constructs which would allow me to make these regions of the huntingtin protein in insect cells. Insect cells were chosen as the expression system of choice as this system generally performs well when trying to make more complicated human proteins.
Most of this lab work was not done by myself but by some seriously talented members of the SGC Toronto biotechnology group – Peter Loppnau (cloner extraordinaire), Ashley Hutchinson (talented insect cell production and test expression scientist) and Alma Seitova (expert in eukaryotic expression systems). Thanks to all of them for their help with this work.
On to the results! As per usual, all of the data I have is uploaded to Zenodo so feel free to browse through and let me know what you think. This is also very preliminary – we will be following this work up with some validation steps shortly.
Most of the designed constructs do not express although there are some clear positive results which is great! Having chatted to other scientist completing similar experiments, this does seem in line with their own experiences. I am now aiming to scale up production of these hits to see whether I can make and purify meaningful quantities of the protein for other experiments AND whether the protein generated is of sufficient quality i.e. is it folded and functional?
All of the constructs are designed with the predicted secondary structure in mind and it does seem that including additional helices or short runs of amino acids either end of a predicted domain boundary does make a huge difference as to whether soluble expression of the protein is achievable in insect cell culture. This suggests that perhaps some of the weaker hits could be optimised further with subtle changes to the N and C-terminal boundaries so I will need to get my thinking cap on to see if I can design something better.
More data to follow soon!