Download Expression and purification of huntingtin domain

Expression and purification of huntingtin domain constructs spanning aa. P80-G428 – 2017/02/06
Aim: to purify mg quantities of soluble huntingtin domain constructs
Rationale: purifying large amounts of protein would allow next phase experiments characterizing the protein
samples to be completed i.e. crystallization experiments for X-ray crystallography, antibody production,
biophysical analysis etc.
Expression system: all proteins were expressed in a baculovirus expression system (BVES) as detailed in
previous uploads: https://zenodo.org/record/57172
NB: all cell culture experiments were completed by Dr. Alma Seitova, head of the Eukaryotic Expression
Platform team at SGC Toronto and her team. Cell culture conditions are as described in accompanying
document BVES_protocols.docx.
Previous experiments: construct spanning residues P80-G428 was successfully purified
https://zenodo.org/record/258581. Buffer condition optimization suggested that more acidic buffers
stabilized the protein further although the sample remained thermally unstable (Tagg < 40 °C)
https://zenodo.org/record/267193.
2nd February 2017:
12 L Sf9 culture of TOC004-A04
was harvested by centrifugation.
Cell pellets were washed in PBS.
Half of the cell paste was then
resuspended in ~ 400 ml Tris
resuspension buffer (50 mM
Tris-HCl pH 8, 500 mM NaCl, 2
mM TCEP, 5 % glycerol – repeat
of the successful conditions)
whilst the other half was
resuspended in ~ 400 ml Hepes
resuspension buffer (50 mM
Hepes pH 7, 500 mM NaCl, 2
mM TCEP, 5 % glycerol – more
acidic conditions to try and improve stability). Samples were flash frozen in liquid nitrogen and stored at -80
°C prior to subsequent purification steps.
Cell suspensions were thawed and rocked at 4 °C for 30 mins with 0.4 % NP-40, 10 U/ml benzonase and 1x
protease inhibitor mix (0.1 mg/ml Aprotinin, 0.1 mg/ml Leupeptin, 0.2 mg/ml Pepstatin A, 0.1 mg/ml E-64).
Suspensions were then diluted to 2 x 250 ml. Cell lysates were clarified by centrifugation at 20,000 xg for 1
hour.
Each clarified lysate was rocked with 5 ml TALON (cobalt-beads) resin at 4 °C for 1 hour (FT). Beads were
washed with 250 ml appropriate resuspension buffer (W1) and then 250 ml appropriate resuspension buffer
supplemented with 15 mM imidazole (W2). Protein was eluted with 20 ml resuspension buffer supplemented
with 300 mM imidazole (E). Samples collected throughout the prep were analysed by 4-20 % Tris-Glycine SDSPAGE.
No obvious band for the protein is seen in the elution fraction on the SDS-PAGE
(expected MW ~ 40.1 kDa). To verify whether protein was expressed in the BVES
production, 3 mL samples (saved by the eukaryotic production team) of the
original culture underwent testX purification as per BVES protocols. Elution of the
sample from cobalt purification were analysed by SDS-PAGE.
The protein is present in these elution samples. Unfortunately this implies that I
“lost” the protein during the purification. The key difference between my
purification method and that of the testX is that it is much slower due to the larger
cell paste volume as well as running two purifications in parallel to compare
different buffer conditions. Due to the low stability of the protein, extended
purification times could cause protein precipitation.
Therefore the production and purification will be repeated with a maximum of 4 L
cell culture, with no other experiments running in parallel to ensure the protein is
taken from cell lysis to gel filtration as quickly as possible.