Figure 3
Purification and characterization of Gg-laforin. A. The chromatogram is of His6-SUMO- tagged Gg-laforin first purified using an IMAC affinity column, and then passed over a HiLoad 16/60 Superdex 200 size exclusion column. Calibration of molecular weight markers is indicated, and ordinates indicate the natural logarithm of molecular weight (Mr). B. The monomer fractions were collected, concentrated, and re-loaded over the same column. The chromatogram shows the results of this second round of size-exclusion chromatography. C. SDS-PAGE stained with Coomassie Blue of fractions from E. coli cells: (U) uninduced cells; (I) induced with IPTG; (P) pelleted/insoluble fraction; (S) soluble fraction; (E) IMAC eluate; and (S200) monomer fraction from Superdex 200 elution. 20 μg of total protein was loaded per lane. His6-SUMO-Gg-laforin runs as a 50 kDa species until removal of the His6-SUMO tag after IMAC elution (indicated by the arrow). Untagged Gg-laforin is predicted to be 36 kDa (indicated by the asterisk). D. Dynamic light scattering was performed on a 1 mg/ml sample of Superdex 200-purified Gg-laforin monomer using a Protein Solutions DynaPro-99 system. Scattering intensity was measured and presented as a fraction of the total protein mass. A single species was detected with a hydrodynamic radius of 2.68 nm, corresponding to a molecular weight of 31.6±14.5 kDa.