Nearly all of my experiments, whether interaction or structural studies, require large (ish) amounts of purified huntingtin protein. This is why a lot of my posts and write ups describe making the huntingtin protein. Over a year into this project, I have now established good protocols for expressing and purifying the protein in large quantities in an insect cell system to high purity and homogeneity.
To date, my large scale purifications make an N-terminally tagged version of the huntingtin protein, meaning that the tag is very close to the exon 1 region (the polyQ stretch) of the protein. I (and the very helpful Ray Truant) have been concerned that this may affect the fold or function of this region of the native protein so Peter Loppnau, an excellent molecular biologist at SGC Toronto, recloned the huntingtin gene into the pBacMam-diex-lic vector with a C-terminal tag instead.
I purified this protein in a large scale experiment from insect cells and you can see my write up here on Zenodo. The yield was EXCELLENT with 2.6 mg of protein from 3 L (as measured at the point of freezing the final sample for storage) and the ratio of aggregate:monomeric protein species was much more in favour of the monomeric protein. I am very keen to repeat this purification experiment to see if this is a random event or whether this new construct is just a lot better at making happy stable protein.
Anyone interested in receiving plasmid or purified protein, please get in touch! I am happy to share. The huntingtin pBacMam vectors will be in Addgene once we have validated them further.