Fig. 4

Production and purification of tag-free hIDS via enterokinase cleavage. (A) Proteolytic cleavage of purified GB1-CBD1-hIDS-su using enterokinase. Purified GB1-CBD1-hIDS-su (2.5 mg) was incubated with enterokinase (50 units) at 25^oC for 16 h. An aliquot of incubated samples together with other controls was separated by SDS-PAGE followed by CBB staining. Lane M, protein size standard; lane NT, non-transformed leaves; lane 3, GB1-CBD1-hIDS-su purified using Ni²⁺-NTA resins; lane 4, enterokinase-treated GB1-CBD1-hIDS-su; lane 5, MCC bead-bound proteins; lane 6, supernatant contains only hIDS-su protein. (B) Quantification of tag-free hIDS-su. The incubated mixture from A was incubated with MCC beads and the supernatant was collected to recover the tag-free hIDS-su. The supernatant was concentrated using Centricon to 4 mL of final volume. The indicated volumes of hIDS-su were separated by SDS-PAGE together the indicated amount of human serum albumin (HSA), followed by staining with CBB.