Fig. 7
Purification of the depolymerase and other soluble TP-84 proteins from TP-84/G. stearothermophilus strR lysates. Protein bands were cut-out and subjected to LC–MS. Bands, corresponding to the depolymerase are marked with arrows. Panel a. 10% SDS-PAGE of purification stages (Protocol 1). Lane M, PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa; Lane 1, TP-84 TCA-precipitated from 350 μl lysate; Lane 2, depolymerase back-extracted from PEI complexes; Lane 3, flow-through from Phosphocellulose P11; Lane 4, combined peak fractions from Q-Sepharose; Lane 5, flow-through from Heparin-Sepharose. Panel b. Final depolymerase size fractionation and concentration (Protocol 1). Lane M, PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa; Lane 1, TP84_26 depolymerase after VivaSpin® Turbo 15 RC concentration. Panel c. Purification of TP-84 proteins using Protocol 2. Lane M, PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa; Lane 1, Q-Sepharose chromatography—fraction 100 mM; Lane 2, 200 mM. Panel d. Purification of TP-84 proteins using modified Protocol 2, containing CM-Sepharose (negative step), used before Q-Sepharose. Lane M, PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa; Lane 1, Q-Sepharose—fraction 500 mM