Small scale and large scale expression and purification of huntingtin protein in
HEK293 mammalian expression system – 2016/11/23
Aim: to generate samples of full-length huntingtin protein, representative of the general
population (Q17) and HD patient population (Q46), from a mammalian expression system.
Rationale: subtle differences in protein fold and structure compared to a baculovirus expression
system (BVES) due to differences in post-translational modifications and available chaperone
machinery should be reconciled by expressing the protein in a recombinant mammalian system.
Expression system: doxycycline-inducible Htt expression in stable cell lines for full-length HTT Q17
and Q46, kindly provided by Prof. Stefan Kochanek and the CHDI Foundation, detailed in this
publication: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4370734/
NB: all cell culture experiments were completed by Dr. Alma Seitova, head of the Eukaryotic
Expression Platform team at SGC Toronto. Cell culture conditions are as described in accompanying
document HEK293_HTT_Methods.pdf which was kindly provided by Prof. Stefan Kochanek, Ulm
University, whose lab generated the cell lines used in this experiment.
Previous test expression experiments completed by Dr. Alma Seitova: All test expression
experiments were completed with 1 x 10 cm petri dish of cells per construct. Media was removed
and cells resuspended in ~5 mL lysis buffer (50 mM Tris-HCl pH8, 500 mM NaCl, 1x Pis with
benzonase) and lysed by 2 freeze-thaw cycles. Clarified lysate was incubated with 50 uL of antiFLAG beads for 2 hours. Beads were washed with 2 x 1 mL wash buffer (50 mM Tris-HCl pH8, 500
mM NaCl). Protein eluted with 30 uL 400 µg/mL FLAG peptide in 50 mM Tris-HCl pH8, 500 mM
NaCl. Elution was mixed with 10 uL of 4 x loading dye and 20 uL run on 4-20 % SDS-PAGE.
1. FLAG purification of full-length hungtingtin (HTT) Q17 from 10 cm petri dish – 8th October
2016
GFP control shows good expression. A faint band is visible for full-length HTT Q17 on the gel at
~350 kDa.
2. FLAG purification of full-length hungtingtin (HTT) Q17/Q46 from 10 cm petri dish – 21st
October 2016
A clear band is visible for full-length HTT Q17 and Q46 on the gel at ~350 kDa.
Large scale purification of full-length HTT Q17 and Q46 from HEK293 cells:
A protocol has been optimized for purifying large scale amounts of full-length huntingtin protein
from BVES. This will be followed for the first pass large scale purification from HEK293 expression
system.
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For both Q length constructs, 8 x 175 cm2 flasks of cells were induced with doxycycline as per
attached protocol to induce expression.
Media was removed and cells resuspended in ~2 x 100 mL lysis buffer for each construct (50
mM Tris-HCl pH8, 500 mM NaCl, 0.1% Tween-20, 1x Pis with benzonase) and lysed by 2 freezethaw cycles – provided to Rachel Harding 2016/11/04.
Experiment started 2016/11/07: Lysates were clarified by centrifugation at 15,000 rpm for 1
hour at 4C.
Clarified lysate was incubated with 2 mL of anti-FLAG beads for 2 hours.
Beads were washed with 2 x 250 mL wash buffer (50 mM Tris-HCl pH8, 500 mM NaCl).
Protein eluted with 2 x 5 mL 400 µg/mL FLAG peptide in 50 mM Tris-HCl pH8, 500 mM NaCl.
Elution fractions were concentrated to ~1 mL using MWCO 100,000 spin concentrators.
Samples of the purification were run on 4-20 % SDS-PAGE for analysis.
FLAG purification shown for full-length HTT – flow through (FT), wash (W) and elution (E).
Very little HTT protein was successfully purified for either polyQ length. These results are
comparable with the first test expression experiment completed by Alma Seitova. However given
the increased number of cells from which the purification was performed used in this instance, I
would have expected much higher yields. It seems likely that there are some issues in terms of
scaling up the cell culture and protein induction – potentially changes in aeration in different flasks,
doxocycline dosing etc.
Next steps: repeat test scale (10 cm2 petri dish) and then follow up with small scale (1 x 175 cm2
flask) expression and purification to validate protein expression in different vessels.