Elsevier

Gene

Volume 178, Issues 1–2, 1996, Pages 107-110
Gene

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

https://doi.org/10.1016/0378-1119(96)00343-5Get rights and content

Abstract

The Igaβ 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 Igaβ 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 Igaβ-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 Igaβ-mediated export pathway. E. coli (dsbA ompT) is highly suitable as a strain for the surface display of recombinant proteins via Igaβ, whether or not they contain cysteine residues.

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    Without the addition of this reducing agent, the autotransporter protein resided in the periplasm and was degraded by proteases located therein [55]. For surface translocation of cysteine-rich passengers, it was shown that it can be helpful to disable the periplasmic oxidoreductase DsbA, which is involved in disulphide bond formation [56]. As in the case of outer membrane proteases, analogous (or also homologous) enzymes have to be identified and possibly knocked out in new host organisms to enable the surface translocation of disulphide bond containing enzymes.

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  • Expression of active human P450 3A4 on the cell surface of Escherichia coli by Autodisplay

    2012, Journal of Biotechnology
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    Secondary structure analysis (TMpred program) of the CYP3A4 sequence suggested that the N-terminal part forms a transmembrane helix, which could interfere with the transport of the enzyme across the outer membrane. It could result in a periplasmic degradation of the passenger fusion protein as has been shown before for the Autodisplay passengers CtxB (Jose et al., 1996) and Aprotinin (Jose and Zangen, 2005). To overcome this possible limitation, the N-terminal 17 amino acids were deleted by a PCR based approach and the resulting truncated CYP3A4 autotransporter fusion protein was analyzed for transport and enzymatic activity as described above.

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1

Present address: EVOTEC BioSystems GmbH, Grandweg 64, D-22529 Hamburg, Germany.

2

Present address: Mucos Pharma, Miraustrasse 17, D-13509 Berlin, Germany.

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