SDS-PAGE analysis of the recombinant protease. a. Total extracts of E. coli Rosetta cells expressing SBcas3.3 or one of its two artificially N-truncated variants (cas516 or cas560). C-: Total extract of induced E. coli Rosetta 2 (DE3) cells transformed with the empty vector pET-30b(+); lanes 1 and 4: total extracts of cells overproducing SBcas3.3 at 25°C (lane 1, cleaved protease, the asterisks indicate the two parts of the protein) or 37°C (lane 4, immature full-length protease); lanes 2 and 3: total extracts of cells overproducing N-truncated forms of the protease (cas516 and cas560). b. Purification of the mature protease. Proteins recovered during different purification steps were separated by SDS–10% polyacrylamide gel electrophoresis and stained with Coomassie Brilliant Blue. Lane 1: total cell extract of induced bacteria overproducing the mature form of SBcas3.3 (the asterisks indicate the two parts of the protein); lane 2: insoluble fraction of the cell extract containing the mature protease; lane 3: soluble fraction obtained after treatment of the insoluble fraction with urea and Triton X-100; lanes 4 and 5: flow-throughs 1 and 2; lane 6: purified protein; MW: protein molecular weight markers.