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

Abstract

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 b y 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 Ecoli 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 pHM7-1OB 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 Bsubtilis secretion apparatus. Under optimized conditions, a level of 4500 U I-' could be obtained in batch cultures.

Bifunctional Role of theTreponema pallidumExtracellular Matrix Binding Adhesin Tp0751

“…Gel-based analysis of heterologously expressed proteases often results in the appearance of multiple protein bands which may be due to degradation by E. coli proteases or autolysis (65,77). The fact that pallilysin formed multiple bands when analyzed via SDS-PAGE following purification from both a lon and ompT protease-deficient E. coli expression strain and an insect secretory protein expression system suggests that the protease may be synthesized as a proprotein which undergoes self-lysis, resulting in cleavage of the disordered proline-rich N-terminal prodomain and generation of the mature active protease.…”

Families and clans of cysteine peptidases

“…Family C l l contains clostripain, an endopeptidase secreted by the anaerobic, grampositive eubacterium Ctostridium histoIyticum, which shows strict specificity for arginyl bonds. The enzyme is synthesised as a precursor that is activated by the removal Of an Nterminal propeptide and also the separation of two chains, with the loss of a nonapeptide, so that the mature enzyme has a light chain (containing the catalytic cysteine) and a heavy chain [44]. Unlike papain, clostripain is calcium-dependent, is more rapidly inactivated by iodoacetamide than by iodoacetate, and is not irreversibly inhibited by E-64 [12].…”

Purification and characterization of a clostripain-like protease from a recombinant Clostridium perfringens culture

“…One unit of hydrolytic activity toward each synthetic substrate was defined as the amount of the enzyme that hydrolysed 1 mmol substrate min 21 at 25 uC. The esterase activities toward Tos-L-arginine ME and Tos-L-lysine ME were determined as described by Witte et al (1994) and Mitchell & Harrington (1970). Protein concentrations were measured using Pierce bicinchoninic acid (BCA) protein assay reagent (Pierce) or Bradford protein assay reagent (Bio-Rad) with BSA as the standard.…”

“…Lysylendopeptidase activity was assayed using benzoyl-DL-lysine pnitroanilide (Bz-DL-lysine pNA; Wako), tosyl-L-lysine methyl ester (Tos-lysine ME; Peptide Institute) and acyl-L-lysine p-nitroanilide (Ac-L-lysine pNA; Bachem). The activities toward the substrates containing p-nitroaniline were determined as described by Witte et al (1994) with slight modifications. Briefly, the assay mixture (0.95 ml) comprising 0.25 M substrate, 50 mM Tris/HCl (pH 7.5), 5 mM DTT and 5 mM CaCl 2 was incubated at 25 uC.…”

“…Furthermore, its unique property in the reverse reaction has been utilized for the syntheses of isosteres (Günther et al, 2000) and biomimetic molecules (Yoo & Kirshenbaum, 2005). Attempts to produce recombinant Clo in heterologous hosts failed to give satisfactory yields, probably due to problems associated with codon bias and secretion (Kim et al, 2007;Witte et al, 1994). Thus, we have addressed the question of whether recombinant Clp can be used as an alternative enzyme; we also discuss the applicability of our method to the purification of recombinant Clo.…”