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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.