Engineered H. sapiens Dynein-2 Constructs

A H. sapiens cytoplasmic dynein-2 motor domain construct containing an N-terminal SNAP_f tag^{19 (link)} was modified to make V1 and V2 constructs. For V1, residues 4196–4201 were changed to sequence WIALNL and residues 4202–4307 (inclusive) were deleted. For V2, residues 2160–2211 (inclusive) in AAA2 were removed, corresponding to exon skipping in V2. Variants were introduced using PCR with Q5 polymerase (New England Biolabs, Ipswich, MA, USA). Dynein-2 constructs were expressed in Spodoptera frugiperda (Sf9) insect cells (Thermo Fisher Scientific, Waltham, MA, USA) using a baculovirus system as previously described.^{19 (link)} Protein purifications were performed at 4 °C. Proteins of interest were eluted by resuspending the resin in TEV buffer, adding 20 μg TEV protease, and incubating the reaction overnight on a roller. TEV-cleaved proteins were separated from the resin using an empty column (Supplementary Methods).