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
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.