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Dandyismus und Popkultur
(2013)
Test environments in sound quality research often significantly differ from human’s everyday world. Hence the claim for ecological validity of such experiments is reasonable, but too vague in its bare formulation. The estimation of ecological validity requires theoretical knowledge about the meaning and the totality of factors influencing everyday listening and their interaction. Therefore the influence of “reality factors” on the sound evaluation of household appliances was investigated within the framework of nine experiments. These tests were performed in four different settings with varied reality degrees. The psychological realism and the perceived face validity of the tests were examined by means of interviews. The results reveal, on the one hand, that stimuli are perceived as more pleasant in everyday-like environments. The analysis of the relative judgments shows, on the other hand, that the influence of the experimental realism is moderated by a number of factors. These are the saliency and meaning of the sounds, differences between the assessed devices, and personality traits. The correlation and regression analysis confirms that the perceived sound character is widely independent from experimental realism. However, there is great empirical evidence, that valid assessments of sound quality require the reconstruction of everyday attention and action processes.
Synthesis and release of the bacterial compatible solute 5-hydroxyectoine in Hansenula polymorpha
(2013)
Ectoine and 5-hydroxyectoine belong to the family of compatible solutes which are known to mainly contribute to the adaptation of the cell to osmotic stress by mediation of a constant turgor. In addition the cell's essential functions are maintained under stress conditions like high salinity, heat or aridity stress. Hansenula polymorpha was engineered to catalyze the transformation of monomeric substrates to 5-hydroxyectoine. For this purpose four genes encoding the enzymes of the 5-hydroxyectoine biosynthesis pathway of Halomonas elongata, EctA, EctB, EctC, and EctD, were inserted into the genome of H. polymorpha. Subsequently the syntheses of ectoine and 5-hydroxyectoine were analyzed and optimized. We showed that H. polymorpha is a suitable system for recombinant 5-hydroxyectoine synthesis in gram per liter scale (2.8 g L⁻¹ culture supernatant, 365 μmol/g dcw) in which almost 100% conversion of ectoine to 5-hydroxyectoine without necessity of high salinity were achieved.
The methylotrophic yeast Hansenula polymorpha has been established as a highly efficient expression system. Strong promoters derived from the methanol metabolism pathway and mitotically stable high-copy integration have made this organism very effective for heterologous protein expression.
To improve the processing of secretory proteins targeted by the MFα prepro sequence, variations in the amino acid composition near the processing site of Kex2p were investigated (Manuscript I). The insertion of Ala-Pro between MFα prepro sequence and protein of interest leads to an improved processing of the signal sequence in case of interleukin-6 (IL-6) and interferon-α 2a (IFNα-2a). Modeling of the transition sequences confirmed a better exposition of the Kex2p-cleavage site in dependency of Ala-Pro insertions. Analysis of the secreted proteins revealed activity of an Ala-Pro specific endoprotease. Partial removal of the N-terminal Ala-Pro dipeptide was also detected during IFNα-2a processing. The putative protease responsible for the processing of Ala-Pro dipeptides was identified by gene deletion. Significant similarities with Saccharomyces cerevisiae Ste13p were identified.
To generally improve productivity in the H. polymorpha system new fusion protein strategies were investigated (Manuscript II). The recombinantly strongly expressed cytosolic ferritin heavy chain (FTH1) was analyzed with regard to its potential as fusion partner for difficult-to-express target proteins. A fragment of the parathyroid hormone (PTH) was chosen to explore whether fusion to FTH1 enhances PTH stability. We could show that the expression level of this fusion protein was at least of the magnitude of that of FTH1 without fusion partner. The solubility of the fusion protein was increased in comparison to FTH1 alone and no degradation of PTH occurred when fused C-terminally to FTH1.
Ectoine and 5-hydroxyectoine belong to the family of compatible solutes which are known to contribute to the adaptation of the cell to osmotic stress by mediation of a constant turgor. H. polymorpha was engineered to express the enzymes of the H. elongata 5-hydroxyectoine biosynthesis pathway (Manuscript III). Expression of the enzymes EctB, EctA, EctC and EctD in H. polymorpha resulted in production of 5-hydroxyectoine in gram per liter scale with almost full conversion of ectoine to 5-hydroxyectoine. We showed that 5-hydroxyectoine synthesis was influenced by feeding of methanol during cultivation. Furthermore, 5-hydroxyectoine synthesis was downregulated by the addition of methionine, lysine or threonine indicating the existence of a feedback regulated aspartate kinase in H. polymorpha.
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.