U. Weydemann et al., HIGH-LEVEL SECRETION OF HIRUDIN BY HANSENULA-POLYMORPHA - AUTHENTIC PROCESSING OF 3 DIFFERENT PREPROHIRUDINS, Applied microbiology and biotechnology, 44(3-4), 1995, pp. 377-385
A DNA sequence coding for a subtype of the hirudin variant HV1 was exp
ressed in the methylotrophic yeast Hansenula polymorpha from a strongl
y inducible promoter element derived from a gene of the methanol metab
olism pathway. For secretion, the coding sequence was fused to the KEX
2 recognition site of three different prepro segments engineered from
the MF alpha 1 gene of Saccharomyces cerevisiae, the glucoamylase (GAM
1) gene of Schwanniomyces occidentalis and the gene for a crustacean h
yperglycemic hormone from the shore crab Carcinus maenas. In all three
cases, correct processing of the precursor molecule and efficient sec
retion of the mature protein were observed. In fermentations on a 10-1
scale of a transformant strain harbouring a MF alpha 1/hirudin-gene f
usion yields in the range of grams per litre could be obtained. The ma
jority of the secreted product was identified as the full-length 65-am
ino-acid hirudin. Only small amounts of a truncated 63-amino- acid pro
duct, frequently observed in S. cerevisiae-based expression systems, c
ould be detected.