Thrombin protease

We used site-directed mutagenesis on a plasmid encoding the amino acid sequence of WT TDP-43 LCD (residues 266–414) with an N-terminal His₆ tag and thrombin cleavage site (MRGSHHHHHHLVPRGS) in a pRSET-B vector to make an analogous plasmid construct for S403D/S404D (2-PM) and S403D/S404D/S409D/S410D (4-PM) TDP-43 LCD. Rosetta Escherichia coli was used to express the proteins, and they were purified as previously described (Babinchak et al. 2019 (link)), except that thrombin protease (Cytiva Life Sciences, Marlborough, Massachusetts) was used to cleave the N-terminal tag after FPLC and before HPLC. The post-FPLC protein was diluted by a factor of 16 into a 20 mM potassium phosphate (pH 6) solution. Immediately following this step, thrombin was added (10 U thrombin:1 mg of uncleaved TDP-43 LCD), and the solution was rocked for 48 h at ambient temperature. The cleaved protein was then concentrated (4-fold), and subsequently mixed with guanidinium hydrochloride such that the final concentration of GuHCl was 5.3 M. The mixture was then rocked for 30 minutes until the solution clarified. Subsequently, the solution was concentrated, buffer-exchanged on HPLC, and lyophilized as previously described (Babinchak et al. 2019 (link)). To determine the concentrations of protein stocks, we employed absorbance at 280 nm using an extinction coefficient of 17990 M⁻¹ cm⁻¹.

VP1 pentamer were produced using plasmid pET15b expression vector encoding BKPyV VP1 mutant amino acid sequences from positions 30–300 with an N-terminal hexahistidine tag (His-tag) and a thrombin cleavage site (BioCat Gmbh). The protein was expressed in E.coli BL21 DE3 by IPTG induction. Proteins were purified first by nickel affinity chromatography using a 5 mL HisTrap FF crude column (Cytiva). After protein sample loading, the column was washed with 20 mM Tris pH 7.5, 250 mM NaCl, 10 mM imidazole and 10% glycerol, and proteins were eluted by applying a gradient of elution buffer composed of 20 mM Tris pH 7.5, 250 mM NaCl, 500 mM imidazole and 10% glycerol. For glycan array analysis, the His-tag was retained while for crystallisation this tag was cleaved with 10 U/mg of thrombin protease (Cytiva) for 24 h at 20°C with agitation. Cleaved and uncleaved pentamers were finally purified by size-exclusion chromatography on a Superdex 200 16/600 column (Cytiva) by eluting protein with 20 mM HEPES pH 7.5 and 150 mM NaCl.