The fl-domain of the Neisseria IgA protease precursor (Iga) provides the essential transport function for the protease across the outer membrane. To investigate the secretion function of the f-domain (Igaf), we engineered hybrid proteins between Iga, and the non-toxic 12 kd cholera toxin B subunit (CtxB) and examined their targeting behaviour in Salmonella typhimurium. We show that CtxB-Igao hybrid proteins integrate into the outer membrane, leading to the exposition of the CtxB moiety on the cell surface. Exposed CtxB can be degraded by externally added proteases like trypsin, but can also be specifically cleaved off from membrane-associated Iga, by purified IgA protease. We further demonstrate that folding of the CtxB moiety at the periplasmic side of the outer membrane interferes with its translocation. Prevention of disulphide-induced folding in periplasmic CtxB renders the protein moiety competent for outer membrane transport. Iga, may be of general interest as an export vehicle for even larger proteins from Gram-negative bacteria.

The C‐terminal domain (Iga beta) of the Neisseria IgA protease precursor is involved in the transport of covalently attached proteins across the outer membrane of Gram‐negative bacteria. We investigated outer membrane transport in Escherichia coli using fusion proteins consisting of an N‐terminal signal sequence for inner membrane transport, the Vibrio cholerae toxin B subunit (CtxB) as a passenger and Iga beta. The process probably involves two distinct steps: (i) integration of Iga beta into the outer membrane and (ii) translocation of the passenger across the membrane. The outer membrane integrated part of Iga beta is the C‐terminal 30 kDa core, which serves as a translocator for both the passenger and the linking region situated between the passenger and Iga beta core. The completeness of the translocation is demonstrated by the extracellular release of the passenger protein owing to the action of the E. coli outer membrane OmpT protease. Translocation of the CtxB moiety occurs efficiently under conditions preventing intramolecular disulphide bond formation. In contrast, if disulphide bond formation in the periplasm proceeds, then translocation halts after the export of the linking region. In this situation transmembrane intermediates are generated which give rise to characteristic fragments resulting from rapid proteolytic degradation of the periplasmically trapped portion. Based on the identification of translocation intermediates we propose that the polypeptide chain of the passenger passes in a linear fashion across the bacterial outer membrane.

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The pathogenic, Gram-negative bacteria, Neisseria gonorrhoeae, Neisseria meningitidis and Haemophilus influenzae, secrete immunoglobulin A1 proteases into their extracellular surroundings. An extraordinary feature in the secretory pathway of these putative virulence factors is a self-directed outer membrane transport step allowing the proteins to be secreted autonomously, even from foreign Gram-negative host cells like Escherichia coli. Here we summarize recent achievements in the understanding of IgA protease outer membrane translocation.

Based on an integrated approach of genetic engineering, fermentation process development, and downstream processing, a fermentative chymotrypsinogen B production process using recombinant Pichia pastoris is presented. Making use of the P. pastoris AOX1-promotor, the demand for methanol as the single carbon source as well as an inducer of protein secretion enforced the use of an optimized feeding strategy by help of on-line analysis and an advanced controller algorithm. By using an experimental system of six parallel sparged column bioreactors, proteolytic product degradation could be minimized while also optimizing starting conditions for the following downstream processing. This optimization of process conditions resulted in the production of authentic chymotrypsinogen at a final concentration level of 480 mg‚L -1 in the whole broth and a biomass concentration of 150 g‚L -1 cell dry weight, thus comprising a space-time yield of 5.2 mg‚L -1 ‚h -1 . Alternatively to the high cell density fermentation approach, a continuous fermentation process was developed to study the effects of reduced cell density toward oxygen demand, cooling energy, and biomass separation. This development led to a process with a highly increased spacetime yield of 25 mg‚L -1 ‚h -1 while reducing the cell dry weight concentration from 150 g‚L -1 in fed-batch to 65 g‚L -1 in continuous cultivation.