FOREIGN GENE-EXPRESSION IN HANSENULA-POLYMORPHA - A SYSTEM FOR THE SYNTHESIS OF SMALL FUNCTIONAL PEPTIDES

Citation
Kn. Faber et al., FOREIGN GENE-EXPRESSION IN HANSENULA-POLYMORPHA - A SYSTEM FOR THE SYNTHESIS OF SMALL FUNCTIONAL PEPTIDES, Applied microbiology and biotechnology, 45(1-2), 1996, pp. 72-79
Citations number
25
Categorie Soggetti
Biothechnology & Applied Migrobiology
ISSN journal
01757598
Volume
45
Issue
1-2
Year of publication
1996
Pages
72 - 79
Database
ISI
SICI code
0175-7598(1996)45:1-2<72:FGIH-A>2.0.ZU;2-L
Abstract
We describe the synthesis and purification of two functional peptides, namely human insulin-like growth factor II (IGF-II) and Xenopus laevi s magainin II in Hansenula polymorpha after their synthesis as hybrid proteins fused to the C terminus of endogenous amine oxidase. The hybr id genes, placed under control of the H. polymorpha alcohol oxidase pr omoter (P-AOX), were integrated into the genomic alcohol oxidase locus , yielding stable production strains. High-level synthesis of the fusi on proteins, exceeding 20% of total cellular protein, was obtained whe n the transformed strains were grown in methanol-limited chemostat cul tures; when expressed by itself, i.e. in the absence of the amine oxid ase gene, IGF-II could not be recovered from crude cell extracts, prob ably as a result of rapid proteolytic degradation. Accumulation in per oxisomes did not significantly affect the IGF-II protein stability whe n expressed in the absence of the carrier protein. Apparently, fusion to the large (+/- 78 kDa) amine oxidase carrier particularly stabilize s the peptides and prevents them from proteolysis. After partial purif ication, the fusion partners were readily separated by factor Xa treat ment.