Streamline chelating

The expression vector pGEX-6P-1 containing exon-1 of HTT and encoding 52 glutamine residues (HTTQ52) was kindly provided by Zoya Ignatova, University of Hamburg, and used to express recombinant HTT for aggregation assays. HTTQ52 with N-terminal GST-tag followed by a polyhistidine tag was expressed in E. coli strain BL21 after induction with 400 μM IPTG at 24 °C for 4 h. After harvesting by centrifugation, cells were disrupted by enzymatic lysis and sonification. The first purification step was carried out through Ni²⁺-chelating chromatography on a Streamline Chelating resin (Streamline Chelating, GE Healthcare Life Sciences). Fractions containing the expression construct were further purified in a second step via a glutathione sepharose resin (Glutathione Sepharose 4FF, GE Healthcare Life Sciences). The removal of glutathione was achieved by dialysis overnight against buffer containing 50 mM Tris/HCl, 150 mM KCl, pH 8.5. Fractions of interest were lyophilized and stored at − 20 °C until usage. The purity of the samples was assessed by SDS-PAGE and mass spectrometry. Protein concentrations were determined using UV absorption at 280 nm.

E. coli strain BLR (DE3) (Novagen, EMD Millipore, USA) was used for expression of AMSP-Fu₃₅. Streamline Chelating and Q-Sepharose FF used for purification of recombinant protein were obtained from GE Healthcare, Sweden. C-8 RP-HPLC column was purchased from Discovery Supelco. Complete Freund’s Adjuvant/Incomplete Freund’s Adjuvant (Sigma Aldrich) and Alhydrogel^(R) (Brenntag, Denmark) adjuvants have been used for immunization of small animals. HRP conjugated anti-mice and anti-rabbit secondary antibodies were purchased from Sigma.