title: Increase of calnexin gene dosage boosts the secretion of heterologous proteins by Hansenula polymorpha.
abstract: The type I membrane protein calnexin is a conserved key component of the quality control mechanism in the endoplasmic reticulum. It functions as a molecular chaperone that monitors the folding state of nascent polypeptides entering the endoplasmic reticulum. Calnexin also behaves as a lectin, as its chaperoning activity involves binding of oligosaccharide moieties present on newly imported glycoproteins. We isolated the calnexin gene (HpCNE1) from the methylotrophic yeast Hansenula polymorpha, and used HpCNE1 expression plasmids for super-transformation of H. polymorpha strains secreting target proteins of biotechnological interest. The elevated dosage of HpCNE1 enhanced secretion of the four proteins tested: three glycoproteins and one unglycosylated product. Secretion of bacterial alginate epimerase AlgE1 was increased threefold on average, and secretion of both human interferon-gamma and fungal consensus phytase twofold. With phytase and AlgE1 this improvement was all the more remarkable, as the secretion level was already high in the original strains (g L(-1) range). The same approach improved secretion of human serum albumin, which lacks N-linked glycans, about twofold. Glycosylation of the pro-MFalpha1 leader may account for the effect of calnexin in this case. Our results argue that cooverexpression of calnexin can serve as a generally applicable tool for enhancing the secretion of all types of heterologous protein by H. polymorpha.
authors.affiliation: ARTES Biotechnology GmbH, Erkrath, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Klabunde
authors.forename: Sebastian
authors.initials: S
authors.lastname: Kleebank
authors.forename: Michael
authors.initials: M
authors.lastname: Piontek
authors.forename: Cornelis P
authors.initials: CP
authors.lastname: Hollenberg
authors.forename: Stephan
authors.initials: S
authors.lastname: Hellwig
authors.forename: Adelheid
authors.initials: A
authors.lastname: Degelmann
mesh_terms: D010832:6-Phytase / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D037281:Calnexin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics; D018628:Gene Dosage; D007371:Interferon-gamma / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D016415:Sequence Alignment; D012709:Serum Albumin / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D001426:Bacterial Proteins; D005656:Fungal Proteins; D011994:Recombinant Proteins; D012709:Serum Albumin; D037281:Calnexin; D007371:Interferon-gamma; D010832:6-Phytase; D002238:Carbohydrate Epimerases