FIG 6.

Refolding, autoproteolysis, and toxicity of Mep72. (A) Shown is a Western blot of Mep72 undergoing autoproteolysis. Denatured His₆-Mep72 was refolded by rapidly diluting the urea denaturant. Samples were incubated for the indicated times before the addition of SDS sample buffer to stop any further reaction. The products were resolved by SDS-PAGE and analyzed by Western blotting. An Mep72 sample incubated for 60 min in 8 M urea (i.e., maintained in a denatured state) was used as a control. (B and C) Alginate retards Mep72 autoproteolysis. (B) Urea-denatured Mep72 was refolded as described for panel A, except that the indicated sugars or sugar polymers were included in the refolding mixture. Reactions were allowed to proceed for 20 min before quenching with SDS sample buffer and resolution by SDS-PAGE. The resulting Coomassie blue-stained gel is shown. The refolding buffers contained Tris-HCl–NaCl alone (Tris) or Tris-HCl–NaCl supplemented with 1% (wt/vol) alginate (Alg), glucose (Glu), arabinose (Ara), sucrose (Suc), maltose (Mal), polygalacturonic acid (PGA), or cellulose (Cel), as indicated. (C) Kinetics of Mep72 autoproteolysis with or without 1% (wt/vol) alginate. His₆-Mep72 was refolded as outlined above. Aliquots of the refolded protein mixture were removed and quenched after a 2-, 10-, and 30-min reaction, as indicated. Shown is a Coomassie blue-stained gel of the products. (D) The metzincin domain and carbohydrate-binding domains of Mep72 are not themselves intrinsically toxic in the absence of BamI. Serial dilutions of E. coli containing plasmid-encoded, IPTG-inducible N-terminal MBP fusions to the metzincin- and carbohydrate (CHO)-binding domains of Mep72 were spotted onto LB-agar plates containing IPTG. The spotted dilutions were allowed to grow for 24 h before being photographed to record cell viability.