High-level secretion of hirudin by Hansenula polymorpha--authentic processing of three different preprohirudins.
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.
Weydemann
U
U
Rhein Biotech GinbH, Düsseldorf, Germany.
Keup
P
P
Piontek
M
M
Strasser
A W
AW
Schweden
J
J
Gellissen
G
G
Janowicz
Z A
ZA
D000595:Amino Acid Sequence; D000818:Animals; D060829:Arthropod Proteins; D003386:Brachyura / Q000235:genetics; D005285:Fermentation; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics; D005087:Glucan 1,4-alpha-Glucosidase / Q000096:biosynthesis / Q000235:genetics; D006629:Hirudins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D007447:Invertebrate Hormones / Q000096:biosynthesis / Q000235:genetics; D000072235:Mating Factor; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics; D010452:Peptide Biosynthesis; D010455:Peptides / Q000235:genetics; D010843:Pichia / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics
D060829:Arthropod Proteins; D005656:Fungal Proteins; D006629:Hirudins; D007447:Invertebrate Hormones; D009419:Nerve Tissue Proteins; D010455:Peptides; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C015947:hyperglycemic hormone, crustacean; D000072235:Mating Factor; D005087:Glucan 1,4-alpha-Glucosidase