Foreign gene expression in Hansenula polymorpha. A system for the synthesis of small functional peptides.
We describe the synthesis and purification of two functional peptides, namely human insulin-like growth factor II (IGF-II) and Xenopus laevis magainin II in Hansenula polymorpha after their synthesis as hybrid proteins fused to the C terminus of endogenous amine oxidase. The hybrid genes, placed under control of the H. polymorpha alcohol oxidase promoter (PAOX), were integrated into the genomic alcohol oxidase locus, yielding stable production strains. High-level synthesis of the fusion proteins, exceeding 20% of total cellular protein, was obtained when the transformed strains were grown in methanol-limited chemostat cultures; when expressed by itself, i.e. in the absence of the amine oxidase gene, IGF-II could not be recovered from crude cell extracts, probably as a result of rapid proteolytic degradation. Accumulation in peroxisomes did not significantly affect the IGF-II protein stability when expressed in the absence of the carrier protein. Apparently, fusion to the large (+/- 78 kDa) amine oxidase carrier particularly stabilizes the peptides and prevents them from proteolysis. After partial purification, the fusion partners were readily separated by factor Xa treatment.
Faber
K N
KN
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), Haren, Netherlands.
Westra
S
S
Waterham
H R
HR
Keizer-Gunnink
I
I
Harder
W
W
Veenhuis
G A
GA
D000595:Amino Acid Sequence; D000818:Animals; D023181:Antimicrobial Cationic Peptides; D001483:Base Sequence; D004274:DNA, Recombinant / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D054806:Magainins; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010452:Peptide Biosynthesis; D010455:Peptides / Q000235:genetics* / Q000302:isolation & purification; D010843:Pichia / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D014170:Transformation, Genetic; D029867:Xenopus Proteins; D014982:Xenopus laevis
D023181:Antimicrobial Cationic Peptides; D004274:DNA, Recombinant; D054806:Magainins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; C053301:magainin 2 peptide, Xenopus; D007335:Insulin-Like Growth Factor II