Ivan Diers

Purification and characterisation of a new hypothalamic satiety peptide, cocaine and amphetamine regulated transcript (CART), produced in yeast

Cocaine and amphetamine regulated transcript (CART) is a newly discovered hypothalamic peptide with a potent appetite suppressing activity following intracerebroventricular administration. When the mature rat CART sequence encoding CART(1–102) was inserted in the yeast expression plasmid three CART peptides could be purified from the fermentation broth reflecting processing at dibasic sequences. None of these corresponded to the naturally occurring CART(55–102). In order to obtain CART(55–102) the precursor Glu‐Glu‐Ile‐Asp‐CART(55–102) has been produced and CART(55–102) was generated by digestion of the precursor with dipeptidylaminopeptidase‐1. All four generated CART peptides have been characterised by N‐terminal amino acid sequencing and mass spectrometry. The CART peptides contain six cysteine residues and using the yeast expressed CART(62–102) the disulphide bond configuration was found to be I–III, II–V and IV–VI. When the four CART peptides were intracerebroventricularly injected in fasted mice (0.1 to 2.0 μg) they all produced a dose dependent inhibition of food intake.

Detection of enzyme activity in fractions collected from free solution capillary electrophoresis of complex samples

Abstract Crude fermentation broth from a fermentation of Aspergillus oryzae was analyzed using free solution capillary electrophoresis (FSCE) in a alkaline running buffer. Fractions as large as possible were collected after FSCE separation and analyzed for alkaline protease activity with Suc-Ala-Ala-Pro-Phe-p-nitroanilide as substrate. Two peaks were isolated; one of them was unknown and therefore was further investigated. After amplification of the activity by incubation with Suc-Ala-Ala-Pro-Phe-p-nitroanilide or casein as substrate, the reaction mixtures were analyzed by FSCE. In this way as little as 3 ng of enzyme were identified as an alkaline protease of the subtilisin family.

Yield improvement of heterologous peptides expressed in yps1-disrupted Saccharomyces cerevisiae strains.

Heterologous protein expression levels in Saccharomyces cerevisiae fermentations are highly dependent on the susceptibility to endogenous yeast proteases. Small peptides, such as glucagon and glucagon-like-peptides (GLP-1 and GLP-2), featuring an open structure are particularly accessible for proteolytic degradation during fermentation. Therefore, homogeneous products cannot be obtained. The most sensitive residues are found at basic amino acid residues in the peptide sequence. These heterologous peptides are degraded mainly by the YPS1-encoded aspartic protease, yapsin1, when produced in the yeast. In this article, distinct degradation products were analyzed by HPLC and mass spectrometry, and high yield of the heterologous peptide production has been achieved by the disruption of the YPS1 gene (previously called YAP3). By this technique, high yield continuous fermentation of glucagon in S. cerevisiae is now possible.

Expression of insulin in yeast: the importance of molecular adaptation for secretion and conversion.

The globular, two...chain and 51 amino acid residue peptide-hormone insulin is produced and secreted by the ~-cellsof the pancreatic islets of Langerhans. Insulin is synthesized as preproinsnlin (110 amino acids). The pre-peptide (signal peptide) is removed upon entrance into the endoplasmic reticulum. Proinsulin folds in the endoplasmic reticulum, is transported to the Goigi apparatus and subsequently processed into the mature insulin molecule that is stored in well-defined storage vesicles (Figure 5.1) (Steiner etaI., 1967, 1986; Dodsonand Steiner, 1998). Proinsulin and insulin have self-assembling properties that play an important role in processing and storage in the J3-cells secretory pathway and both associate to dimers and in the presence of zinc these further assemble into hexamers (Dodson and Steiner, 1998). In the late Golgi apparatus proinsulin is targeted to acidifying secretory granules and conversion ofproinsulin to insulin occurs by removal of the C-peptide by cleavage at dibasic processing sites by the endoproteases PC3 (or PCl) and pe2 (mammalian

Yeast cell permeabilizing β‐1,3‐Glucanases: A tool for the integration of downstream processes and metabolic engineering applications to yeast

In this article, we consider the impact on downstream process design resulting from the use of metabolically engineered yeast strains. We address the issue of how manipulation of cell wall permeability can improve the release and subsequent recovery of heterologous products produced in yeast.