Knud Vad

A removable spacer peptide in an α-factor-leader/insulin precursor fusion protein improves processing and concomitant yield of the insulin precursor in Saccharomyces cerevisiae

An alpha-factor leader/insulin precursor fusion protein was produced in Saccharomyces cerevisiae and metabolically labeled in order to analyse the efficiency of maturation and secretion. A substantial fraction of the secreted material was found in a hyperglycosylated unprocessed form, indicating incomplete Kex2p endopeptidase maturation. Introduction of a spacer peptide (EAEAEAK) after the dibasic Kex2p site, creating a N-terminal extension of the insulin precursor, greatly increased the Kex2p catalytic efficiency and the fermentation yield of insulin precursor. The N-terminal extension features a Lys to allow subsequent proteolytic removal by trypsin or the Achromobacter lyticus Lys-specific protease. Dipeptidyl aminopeptidase A (DPAPA) activity removing Glu-Ala dipeptides from the extension was inhibited by adding a Glu N-terminally to the extension. Unexpectedly, this modified N-terminal extension (EEAEAEAK) was partially cleaved after the Lys during fermentation. This monobasic proteolytic activity was demonstrated to be associated with Yap3p. Yap3p cleavage could be prevented by insertion of a Pro before the Lys (EEAEAEAPK).

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.

Accumulation of defence-related transcripts and cloning of a chitinase mRNA from pea leaves (Pisum sativum L.) inoculated with Ascochyta pisi Lib.

Abstract The race specific resistance of pea to Ascochyta pisi Lib. was shown to be exhibited as a hypersensitive response associated with the production of polyphenolic substances in epidermal and mesophyll cells. The levels of transcripts representing a pathogenesis-related (PR) protein (chitinase) and an enzyme of phytoalexin biosynthesis (chalcone synthase) were shown to accumulate more rapidly during the hypersensitive response than during lesion development in the compatible interaction. A full-length (1143 bp) cDNA sequence of a pea chitinase (EC 3.2.1.14) (coding for an approx. 34 500 Da protein) was deduced by combining the overlapping sequences of three clones obtained following PCR amplification of cDNA prepared from mRNA isolated 24 h after inoculation of pea leaves with Ascochyta pisi. The combined sequences were identified as a class I chitinase corresponding to the basic A1-chitinase enzyme previously isolated from pea leaves (Vad et al., Planta, 184 (1991) 24–29). Like class III and IV chitinases, the pea sequence differs from other class I chitinases in the absence of a hydrophobic C-terminal domain.

Turoctocog alfa (NovoEight®) – from design to clinical proof of concept

Turoctocog alfa (NovoEight®) is a recombinant factor VIII (rFVIII) with a truncated B‐domain made from the sequence coding for 10 amino acids from the N‐terminus and 11 amino acids from the C‐terminus of the naturally occurring B‐domain. Turoctocog alfa is produced in Chinese hamster ovary (CHO) cells without addition of any human‐ or animal‐derived materials. During secretion, some rFVIII molecules are cleaved at the C‐terminal of the heavy chain (HC) at amino acid 720, and a monoclonal antibody binding C‐terminal to this position is used in the purification process allowing isolation of the intact rFVIII. Viral inactivation is ensured by a detergent inactivation step as well as a 20‐nm nano‐filtration step. Characterisation of the purified protein demonstrated that turoctocog alfa was fully sulphated at Tyr346 and Tyr1664, which is required for optimal proteolytic activation by thrombin. Kinetic assessments confirmed that turoctocog alfa was activated by thrombin at a similar rate as seen for other rFVIII products fully sulphated at these positions. Tyr1680 was also fully sulphated in turoctocog alfa resulting in strong affinity (low nm Kd) for binding to von Willebrand factor (VWF). Half‐lives of 7.2 ± 0.9 h in F8‐KO mice and 8.9 ± 1.8 h haemophilia A dogs supported that turoctocog alfa bound to VWF after infusion. Functional studies including thromboelastography analysis of human haemophilia A whole blood with added turoctocog alfa and effect studies in mice bleeding models demonstrated a dose‐dependent effect of turoctocog alfa. The non‐clinical data thus confirm the haemostatic effect of turoctocog alfa and, together with the comprehensive clinical evaluation, support the use as FVIII replacement therapy in patients with haemophilia A.