HETEROLOGOUS EXPRESSION OF THE CLOSTRIPAIN GENE FROM CLOSTRIDIUM-HISTOLYTICUM IN ESCHERICHIA-COLI AND BACILLUS-SUBTILIS - MATURATION OF THECLOSTRIPAIN PRECURSOR IS COUPLED WITH SELF-ACTIVATION

Clostripain-specific antibodies were used to analyse the maturation of clostripain prepro-enzyme and core protein heterologously synthesized in Escherichia coli and Bacillus subtilis. Core protein purified from E. coli cells harbouring plasmid pHM3-23 underwent calcium-dependent, self-triggered maturation. Concomitantly, the inactive form of the en zyme was converted into an active form, demonstrating the self-activat ion capacity of the clostripain core protein. As judged from Western b lot analysis, the major portion of the protein in E. coli was degraded , presumably by the activated clostripain. The enzyme was not exported to the E. coli periplasm, either by use of the putative Clostridium h istolyticum signal peptide or by use of the E. coli OmpA signal peptid e. Therefore, the Cram-positive micro-organism B. subtilis was chosen as an alternative host for the expression of the prepro-enzyme and the core protein. BR 151 cells harbouring pHM7-10B secreted clostripain p recursor to the growth medium and matured subsequently to the active e nzyme. As only a small amount of activity was detected intracellularly , the putative C. histolyticum signal peptide was efficiently recogniz ed by the B. subtilis secretion apparatus. Under optimized conditions, a level of 4500 U l(-1) could be obtained in batch cultures.