| Title: | Improved processing of secretory proteins in Hansenula polymorpha by sequence variation near the processing site of the alpha mating factor prepro sequence |
| Author(s): | Eilert E; Rolf T; Heumaier A; Hollenberg CP; Piontek M; Suckow M; |
| Address: | "ARTES Biotechnology GmbH, Elisabeth-Selbert-Str. 9, 40764 Langenfeld-Rheinland, Germany. e.eilert@artes-biotechnology.com" |
| DOI: | 10.1016/j.jbiotec.2012.08.024 |
| ISSN/ISBN: | 1873-4863 (Electronic) 0168-1656 (Linking) |
| 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 MFalpha 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 MFalpha 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 MFalpha 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 MFalpha prepro-interferon alpha-2a (IFNalpha-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 IFNalpha-2a led to correct processing and elimination of all high molecular weight isoforms observed in earlier experiments" |
| Keywords: | Amino Acid Sequence Amino Acids/genetics/metabolism Biotechnology/*methods Fungal Proteins/*genetics/metabolism Genetic Variation Interferon alpha-2 Interferon-alpha/genetics/metabolism Interleukin-6/genetics/metabolism Mating Factor Molecular Sequence Da; |
| Notes: | "MedlineEilert, Eva Rolf, Theresa Heumaier, Andreas Hollenberg, Cornelis P Piontek, Michael Suckow, Manfred eng 2P41RR001081/RR/NCRR NIH HHS/ 9P41GM103311/GM/NIGMS NIH HHS/ Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Netherlands 2012/09/18 J Biotechnol. 2013 Aug 20; 167(2):94-100. doi: 10.1016/j.jbiotec.2012.08.024. Epub 2012 Sep 7" |
