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High-level secretion of hirudin by Hansenula polymorpha —authentic processing of three different preprohirudins

  • Biotechnology
  • Original Paper
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Abstract

A DNA sequence coding for a subtype of the hirudin variant HV1 was expressed in the methylotrophic yeast Hansenula polymorpha from a strongly inducible promoter element derived from a gene of the inducible promoter element derived from a gene of the methanol metabolism pathway. For secretion, the coding sequence was fused to the KEX2 recognition site of three different prepro segments engineered from the MFα1 gene of Saccharomyces cerevisiae, the gluco-amylase (GAM1) gene of Schwanniomyces occidentalis and the gene for a crustacean hyperglycemic hormone from the shore crab Carcinus maenas. In all three cases, correct processing of the precursor molecule and efficient secretion of the mature protein were observed. In fermentations on a 10-1 scale of a transformant strain harbouring a MFα1/hirudin-gene fusion yields in the range of grams per litre could be obtained. The majority of the secreted product was identified as the full-length 65-amino-acid hirudin. Only small amounts of a truncated 63-amino- acid product, frequently observed in S. cerevisiae-based expression systems, could be detected.

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Authors and Affiliations

  1. Rhein Biotech GmbH, Eichsfelder Str. 11, D-40595, Düsseldorf, Germany

    U. Weydemann, P. Keup, M. Piontek, A. W. M. Strasser, G. Gellissen & Z. A. Janowicz

  2. BASF AG, D-67056, Ludwigshafen, Germany

    J. Schweden

Authors
  1. U. Weydemann
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  2. P. Keup
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  3. M. Piontek
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  4. A. W. M. Strasser
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  5. J. Schweden
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  6. G. Gellissen
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  7. Z. A. Janowicz
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Weydemann, U., Keup, P., Piontek, M. et al. High-level secretion of hirudin by Hansenula polymorpha —authentic processing of three different preprohirudins. Appl Microbiol Biotechnol 44, 377–385 (1995). https://doi.org/10.1007/BF00169932

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  • DOI: https://doi.org/10.1007/BF00169932

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