Clostripain-specific antibodies were used to analyse the maturation of clostripain prepro-enzyme and core protein heterologously synthesized in and Core protein purified from cells harbouring plasmid pHM3-23 underwent calcium-dependent, self-triggered maturation. Concomitantly, the inactive form of the enzyme was converted into an active form, demonstrating the self-activation capacity of the clostripain core protein. As judged from Western blot analysis, the major portion of the protein in was degraded, presumably by the activated clostripain. The enzyme was not exported to the periplasm, either by use of the putative signal peptide or by use of the OmpA signal peptide. Therefore, the Gram-positive micro-organism 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 precursor to the growth medium and matured subsequently to the active enzyme. As only a small amount of activity was detected intracellularly, the putative signal peptide was efficiently recognized by the secretion apparatus. Under optimized conditions, a level of 4500 UI could be obtained in batch cultures.


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