Human serum albumin prepared by blood fractionation for clinical purposes was found to degrade when stored at or above 30°C. Mass spectrometry and N-terminal sequencing of the protein identified degradation corresponding to the loss of the first two residues, aspartic acid and alanine. The reaction was shown to be dependent upon temperature and the N-terminal a-amino group. In addition, comparison with serum albumins derived from other species showed that the instability of the N-terminus was specific to the human albumin sequence. An intact aspartyl-alanyl dipeptide, purified from degraded albumin solutio'ns, differed substantially from a synthetic dipeptide on amino acid analysis, N-terminal sequencing and NIMR. It is suggested that the released dipeptide may be cyclic, implying a novel cleavage mechanism.

Recombinant human platelet-derived endothelial cell growth factor, expressed in the yeast Saccharomyces cerevisiae was purified to greater than 98 % purity by anion-exchange and hydroxyapatite chromatography. It was shown to possess thymidine phosphorolytic activity in vitro (pH optimum, pH 5.3; K,, 0.11 mM; V, , , 12.5 mmol min-' mg-'; turnover number, 9.4 s -I ) . Covalent modification simultaneously inhibited the enzymatic and mitogenic properties of the protein, while interaction with a cell-surface receptor was not required to stimulate mitogenesis. Purified Escherichia coli thymidine phosphorylase was also mitogenic toward endothelial cells. It is proposed that platelet-derived endothelial cell growth factor is human thymidine phosphorylase which promotes endothelial cell proliferation by reducing thymidine levels that would otherwise be inhibitory to endothelial cell growth.

BackgroundAnimal-free recombinant proteins provide a safe and effective alternative to tissue or serum-derived products for both therapeutic and biomanufacturing applications. While recombinant insulin and albumin already exist to replace their human counterparts in cell culture media, until recently there has been no equivalent for serum transferrin.ResultsThe first microbial system for the high-level secretion of a recombinant transferrin (rTf) has been developed from Saccharomyces cerevisiae strains originally engineered for the commercial production of recombinant human albumin (Novozymes' Recombumin® USP-NF) and albumin fusion proteins (Novozymes' albufuse®). A full-length non-N-linked glycosylated rTf was secreted at levels around ten-fold higher than from commonly used laboratory strains. Modification of the yeast 2 μm-based expression vector to allow overexpression of the ER chaperone, protein disulphide isomerase, further increased the secretion of rTf approximately twelve-fold in high cell density fermentation. The rTf produced was functionally equivalent to plasma-derived transferrin.ConclusionsA Saccharomyces cerevisiae expression system has enabled the cGMP manufacture of an animal-free rTf for industrial cell culture application without the risk of prion and viral contamination, and provides a high-quality platform for the development of transferrin-based therapeutics.

We report the cloning of the Aspergillus niger glucose oxidase gene and its use to elevate glucose oxidase productivity in A. niger by increasing the gene dosage. In addition, the gene has been introduced into A. nidulans where it provides the novel capacity to produce glucose oxidase. A plasmid, in which DNA encoding the mature form of glucose oxidase was preceded by a Saccharomyces cerevisiae secretion signal, effected high-level production of extracellular glucose oxidase in this yeast.

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