Fig. 3

Purification for active form hIDS-su from total protein extracts. (A) Purification of GB1-CBD1-hIDS-su. Total protein extracts (TPE) from infiltrated leaves were purified using Ni²⁺-NTA resin. Proteins in the fractions from the purification process were separated by SDS-PAGE followed by CBB staining (left panel) and Western blot analysis (right panel). In the CBB staining, Lane M, protein size standard; lane NT, non-transformed leaves; lane 3, total protein extract of hIDS; lane 4–5, flow though samples; lane 6–7, washing-off solutions; lane 8, eluents; lane 9, proteins bound to Ni²⁺-NTA after elution using 500 mM imidazole. (B) Elution profile of Ni²⁺-NTA purified GB1-CBD1-hIDS-su on size exclusion column. Purified proteins using Ni²⁺-NTA affinity column chromatography were loaded to the size exclusion column and fractions were collected. (C) Analysis of fractions from size exclusion chromatography of GB1-CBD1-hIDS-su. The indicated fractions (40–53) were separated by SDS-PAGE, followed by CBB staining. (D) Quantification of purified GB1-CBD1-hIDS-su. Fractions from 40 to 53 of size exclusion chromatography for GB1-CBD1-hIDS-su were combined and concentrated to 4 mL. 1, 2, 4 or 8 µL of purified GB1-CBD1-hIDS-su were separated by SDS-PAGE along with human serum albumin (HSA, 0.25 to 2.0 µg) as reference. The concentration was estimated by comparing HSA.