Heterologous expression of the clostripain gene from Clostridium histolyticum in Escherichia coli and Bacillus subtilis: maturation of the clostripain 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 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 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 histolyticum signal peptide or by use of the E. coli OmpA signal peptide. Therefore, the Gram-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 precursor to the growth medium and matured subsequently to the active enzyme. As only a small amount of activity was detected intracellularly, the putative C. histolyticum signal peptide was efficiently recognized by the B. subtilis secretion apparatus. Under optimized conditions, a level of 4500 U I-1 could be obtained in batch cultures.
Witte
V
V
Weissheimer Research Laboratory, Andernach, Germany.
Wolf
N
N
Diefenthal
T
T
Reipen
G
G
Dargatz
H
H
D001412:Bacillus subtilis / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D003013:Clostridium / Q000235:genetics*; D003546:Cysteine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis
D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; C019913:clostripain