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

• Published in: Applied microbiology and biotechnology Vol. 44; no. 3/4; pp. 377 - 385
• Main Authors: Weydemann, U, Keup, P, Piontek, M, Strasser, A.W.M, Schweden, J, Gellissen, G, Janowicz, Z.A
• Format: Journal Article
• Language: English
• Published: Berlin Springer 01-12-1995
• Subjects: Amino Acid Sequence; Animals; Arthropod Proteins; Biological and medical sciences; Biological Sciences; Biotechnology; Brachyura - genetics; Fermentation; Fundamental and applied biological sciences. Psychology; Fungal Proteins - biosynthesis; Fungal Proteins - genetics; Genetic engineering; Genetic technics; Glucan 1,4-alpha-Glucosidase - biosynthesis; Glucan 1,4-alpha-Glucosidase - genetics; Hansenula polymorpha; Hirudins - biosynthesis; Hirudins - genetics; Hirudins - metabolism; Invertebrate Hormones - biosynthesis; Invertebrate Hormones - genetics; Mating Factor; Methods. Procedures. Technologies; Modification of gene expression level; Molecular Sequence Data; Nerve Tissue Proteins - biosynthesis; Nerve Tissue Proteins - genetics; Peptide Biosynthesis; Peptides - genetics; Pichia - metabolism; Promoter Regions, Genetic; Protein Precursors - metabolism; Protein Processing, Post-Translational; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Saccharomyces cerevisiae - genetics; Recombinant microorganism; Yeast; Molecular processing; Leader sequence; Secretion; Gene expression; Fermentation; Precursor; Fungi; Plasmid; Ascomycetes; Recombinant protein; Hansenula polymorpha; Hybrid gene; Thallophyta
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/BF00169932

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 methanol metabolism pathway. For secretion, the coding sequence was fused to the KEX2 recognition site of three different prepro segments engineered from the MF alpha 1 gene of Saccharomyces cerevisiae, the glucoamylase (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 MF alpha 1/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.