Whilst the purification of the full-length huntingtin protein from the insect cell system we have working in the lab, gives high yields so plenty of protein to do experiments with, the protein is not made in a way which 100 % represents how it is made in humans. Human cells modify the protein with chemical entities called post-translational modifications which can be crucial to proper fold and function of the protein being generated.
To investigate this further, I am using a stable human cell line (HEK293 cells) in which I can switch on a copy of the huntingtin gene for expression by adding tetracycline to the culture. 2 of these cell lines, for expression of huntingtin Q17 and Q46, were kindly provided to us by Stefan Kochanek and Bin Huang. The cell lines give us comparable yields in our hands but the yield is much lower than using the insect cell production system as the cell mass is much lower. I am hoping to look at these samples by EM in my upcoming visit to Oxford, perhaps these samples are more homogenously folded?
All of the data for these experiments can be found on Zenodo.
Alma Seitova kindly grew 10 T-160 flasks of HEK293 cells for each huntingtin construct for me!