title: Improved processing of secretory proteins in Hansenula polymorpha by sequence variation near the processing site of the alpha mating factor prepro sequence.
abstract: The literature as well as databases are ambiguous about the exact start of human interleukin-6 (IL-6)--three possibilities for the initiation of the mature protein are described. These three variants of IL-6, different in the exact initiation of the mature protein (A28, P29, or V30), were expressed in Hansenula polymorpha using the Saccharomyces cerevisiae MFα prepro sequence instead of the homologous pre sequence. All three IL-6 variants were secreted but the processing by the Kex2 protease showed significant differences. V30-IL-6 showed correctly processed material but also a molecule species of higher molecular weight indicating incomplete processing of the MFα pro peptide. P29-IL-6 did not yield any correctly processed IL-6, instead only the unprocessed pro form was found in the culture supernatant. Only A28-IL-6 led to 100% correctly processed material. N-terminal sequencing of this material revealed a start at V30--obviously the first two amino acids (Ala28-Pro29) have been removed by a so far unknown protease. Thus expression of both A28-IL-6 and V30-IL-6 as MFα prepro fusion proteins resulted in the very same mature V30-IL-6, however, the ratio of correctly processed molecules was significantly higher in the case of A28-IL-6. The expression of an MFα prepro-interferon α-2a (IFNα-2a) fusion protein in H. polymorpha leads to about 50% correctly processed molecules and 50% misprocessed forms which contain part of the pro peptide at the N-termini. The insertion of A28 and P29 of IL-6 between the pro peptide and the start of the mature IFNα-2a led to correct processing and elimination of all high molecular weight isoforms observed in earlier experiments.
authors.affiliation: ARTES Biotechnology GmbH, Elisabeth-Selbert-Str. 9, 40764 Langenfeld-Rheinland, Germany. e.eilert@artes-biotechnology.com
authors.forename: Eva
authors.initials: E
authors.lastname: Eilert
authors.forename: Theresa
authors.initials: T
authors.lastname: Rolf
authors.forename: Andreas
authors.initials: A
authors.lastname: Heumaier
authors.forename: Cornelis P
authors.initials: CP
authors.lastname: Hollenberg
authors.forename: Michael
authors.initials: M
authors.lastname: Piontek
authors.forename: Manfred
authors.initials: M
authors.lastname: Suckow
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D001709:Biotechnology / Q000379:methods*; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D014644:Genetic Variation; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000235:genetics / Q000378:metabolism; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D000072235:Mating Factor; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D043484:Proprotein Convertases / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000596:Amino Acids; D005656:Fungal Proteins; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D015850:Interleukin-6; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D000072235:Mating Factor; D043484:Proprotein Convertases; C058751:KEX2 protein, S cerevisiae