Bl21 de3 plyss cells

Nanobodies were cloned in pXAP100 vector as already described^{16 (link)}. pXAP100 is similar to pMES4 (genbank GQ907248) but contains a C-terminal His₆-cMyc tag and allows cloning of the VHH repertoire via SfiI-BstEII restriction sites. Twin-Strep nanobodies were design as follow: the synthetic gene encoding full-length T8 nanobody fused to a C-terminal cleavage site for human rhinovirus 3 C (P3C, LEVLFQGP), a cMyc tag (EQKLISEEDL) and a Twin-Strep-tag (WSHPQFEKGGGSGGGSGGSAWSHPQFEK) instead of the His₆-cMyc tag was synthesized by Eurofins Genomics and then recloned into pXAP100 vector using NotI/EcoRV restrictions sites. Then, the modified vector was digested with Sfi/NotI to allow insertion of nanobodies T2a, T4, T27, G3a, or D12 in frame with the P3C-cMyc-Twin-Strep sequence. All constructs were verified by sequencing (Eurofins Genomics). Nanobody expression and purification were performed as previously described^{16 (link)}. Briefly, nanobodies were produced in Escherichia coli (BL21(DE3) pLysS cells, Millipore), purified from the periplasmic extract via either HisPur Ni-NTA resin (ThermoScientific) or Strep-Tactin XT Superflow resin (iba LifeScience) followed by a size exclusion chromatography on a Superdex 200 Increase 10/300 GL (GE Healthcare) equilibrated in 20 mM HEPES pH 7.5, 150 mM NaCl, and 10% (w/v) glycerol.

Buffers and l-His were purchased from Fisher Scientific. Except if specified otherwise, all other chemicals were purchased from Sigma-Aldrich. Ni-NTA resin and BL21(DE3)pLysS cells were purchased from Millipore. Complete EDTA-Free protease inhibitor was purchased from Roche.

Similar to studies performed previously using coexpression of the S. pombe NatB with αSyn in E. coli to produce an N-terminally acetylated αSyn (57 (link), 58 , 65 (link)), both HttQ25-SUMO-6xHis and HttQ44₄-SUMO-6xHis were co-expressed by co-transformation of BL21(DE3) pLysS cells (Millipore) with the SpNatA plasmid. Cotransformed cells were grown in LB media (Millipore) at 37 °C to an A_600nm of ∼0.5 to 0.6 and induced by addition of 0.5 mM IPTG at 16 °C for 16 h. All subsequent purification steps were carried out at 4 °C and are identical to the protocol implemented for the unmodified Htt proteins.