Huntingtin nanobody expression and purification (2017/11/24)

Recently, I was kindly provided with nanobody constructs which binds the proline rich region of huntingtin exon 1 (the first 90 amino acids) by Ray Truant (McMaster University). Nanobodies are small antibody like molecules containing just a single Ig domain which have high affinity for a specific protein and ideally a specific part or motif of that protein. The nanobody in these experiments might be useful to identify the extreme N-terminus or start of the huntingtin protein molecule in our EM envelope or to compete with binding of other N-terminal huntingtin binders.

 

We used the constructs provided by Dr Truant to generate our own versions which would allow us to make large amounts of the nanobody in an E. coli expression system. Recently, Mani Ravichandran, a technician at the SGC, successfully expressed and purified the nanobody and managed to purify almost 5 milligrams of pure protein. You can read all about these experiments on Zenodo. Next I hope to use the nanobody to see if I can 1) see binding to the recombinant full-length huntingtin samples I am making 2) compete with DNA oligo binding.

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