Human chymotrypsinogen B production with Pichia pastoris by integrated development of fermentation and downstream processing. Part 1. Fermentation.

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 space-time 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.

Absence of periplasmic DsbA oxidoreductase facilitates export of cysteine-containing passenger proteins to the Escherichia coli cell surface via the Iga beta autotransporter pathway.

The Iga beta autotransporter function of IgA1 protease from Neisseria gonorrhoeae was assessed in Escherichia coli using the Vibrio cholerae toxin B subunit (CtxB) as a heterologous passenger. N-terminal fusions with Iga beta of native CtxB or mutant CtxB protein containing no cysteines were constructed and analysed in isogenic E. coli mutants carrying defects in either or both the ompT (outer membrane protease T) and dsbA (periplasmic disulfide oxidoreductase) determinants. While export of the cystein-less CtxB passenger was independent of the dsbA genotype, the native CtxB passenger was properly translocated across the outer membrane only in the dsbA mutant background. This effect was consistent in the presence and in the absence of the OmpT protease which rather determined the release of surface-bound CtxB into the medium. Therefore, in agreement with previous observations Iga beta-dependent protein secretion requires an unfolded conformation of the passenger domain and can be blocked by disulfide loop formation in the presence of DsbA. Since DsbA acts in the periplasm, this provides evidence for a periplasmic intermediate in the Iga beta-mediated export pathway. E. coli (dsbA ompT) is highly suitable as a strain for the surface display of recombinant proteins via Iga beta, whether or not they contain cysteine residues.

C-terminal glycine-histidine tagging of the outer membrane protein Iga beta of Neisseria gonorrhoeae.

A glycine-histidine tag (Gly3His6) was added to the C-terminus of a fusion protein consisting of the cholera toxin B-subunit (CtxB) and the IgA protease beta-domain (Iga beta). The aim was to facilitate single-step purification and to create a suitable tool for kinetic and structural studies on Iga beta-driven protein translocation across the outer membrane of Gram-negative bacteria. We demonstrate that the glycine-histidine tag does not interfere with the assembly of Iga beta in the outer membrane and that the translocator function of the modified Iga beta is maintained. The applicability of the new construct for the dissection of the Iga beta mediated translocation process and general aspects of C-terminal histidine tagging of outer membrane proteins are discussed.

The secretion pathway of IgA protease-type proteins in gram-negative bacteria.

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.

Characterization of the Neisseria Iga beta-core. The essential unit for outer membrane targeting and extracellular protein secretion.

Extracellular transport of Neisseria IgA proteases across the bacterial outer membrane is accomplished by the translocation function contained within the C-terminal Iga beta domain of IgA protease precursor proteins. Recently, we reported that Iga beta from N. gonorrhoeae MS11 (Val1097 to Phe1505), fused to a periplasmic passenger protein, facilitated its transport across the outer membrane, leading to surface exposure of the passenger. In the present work we show, by systematic N-terminal truncation of Iga beta, that the functional and structural unit, termed Iga beta-core, corresponds to the C-terminal approximately 274 amino acid residues (Ser1231 to Phe1505). This minimal region retains all the essential features necessary for the translocation of an N-terminally attached passenger across the outer membrane of Escherichia coli, and for its own correct integration into the outer membrane, even in the absence of a passenger protein. The membrane-integrated Iga beta-core constitutes a conserved entity found in the C-terminal regions of Iga beta domains of different N. gonorrhoeae, N. meningitidis and Haemophilus influenzae strains. In contrast, the surface-exposed N termini of the Iga beta domains vary in size and sequence. Based on secondary structure predictions, the key structural feature of the core is a beta-barrel (amphipathic, antiparallel transmembrane beta-strands, interspersed by hairpin turns and loops) which is common to many integral outer membrane proteins of Gram-negative bacteria. We propose that the core has been conserved in evolution, to provide a selective outer membrane export channel for covalently attached polypeptides.

Sequence-specific cleavage of protein fusions using a recombinant Neisseria type 2 IgA protease.

Sequence-specific enzymatic cleavage of protein fusions is an important application in recombinant protein technology. We have used the Neisseria type 2 IgA protease (EC 3.4.24.13), produced and secreted by Escherichia coli host cells, for efficiently processing polypeptides at authentic or engineered target sites. In different substrates, the microbial protease specifically cleaves the peptide bond distal to the second Pro residue of the sequence Yaa-Pro-/-Xaa-Pro, where Yaa stands for Pro (or rarely for Pro in combination with Ala, Gly or Thr) and Xaa stands for Thr, Ser or Ala. Highly specific proteolysis has been obtained not only with soluble and purified protein fusions but also with insoluble aggregates derived from cytoplasmic inclusion bodies. The sequence-specificity and simple production of the recombinant IgA protease make it a versatile tool for the in vitro processing of recombinant proteins.

Selective extracellular release of cholera toxin B subunit by Escherichia coli: dissection of Neisseria Iga beta-mediated outer membrane transport.

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.

Extracellular transport of cholera toxin B subunit using Neisseria IgA protease beta-domain: conformation-dependent outer membrane translocation.

The beta-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 beta-domain (Iga beta), we engineered hybrid proteins between Iga beta and the non-toxic 12 kd cholera toxin B subunit (CtxB) and examined their targeting behaviour in Salmonella typhimurium. We show that CtxB-Iga beta 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 beta 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 beta may be of general interest as an export vehicle for even larger proteins from Gram-negative bacteria.