Comparative studies of recombinant human albumin and human serum albumin derived by blood fractionation.

Recombinant human albumin produced in the yeast Saccharomyces cerevisial and human serum albumin derived from blood fractionation were compared by a series of analytical techniques. These demonstrated that the two proteins were equivalent structurally. However, differences observed in some of the assays indicated that the recombinant product had lower levels of structural heterogeneity than the blood-derived protein.

Site-specific N-terminal auto-degradation of human serum albumin.

Human serum albumin prepared by blood fractionation for clinical purposes was found to degrade when stored at or above 30 degree 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 alpha-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 solutions, differed substantially from a synthetic dipeptide on amino acid analysis, N-terminal sequencing and NMR. It is suggested that the released dipeptide may be cyclic, implying a novel cleavage mechanism.

Thymidine phosphorylase activity of platelet-derived endothelial cell growth factor is responsible for endothelial cell mitogenicity.

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; Km, 0.11 mM; Vmax, 12.5 mmol min-1 mg-1; turnover number, 9.4 s-1). 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.

Expression of the Aspergillus niger glucose oxidase gene in A. niger, A. nidulans and Saccharomyces cerevisiae.

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.

Production of recombinant human serum albumin from Saccharomyces cerevisiae.

Human serum albumin has been constitutively expressed in a Saccharomyces cerevisiae brewing yeast. After cell growth and disruption the product was associated with the insoluble fraction and represented approximately 1% of total cell protein. After the cell debris was extensively washed, the albumin was solubilized with 8 M urea and 28 mM 2-mercaptoethanol in 50 mM sodium carbonate buffer, pH 10. The denatured albumin was refolded by dialysis and further purified by anion exchange and gel filtration chromatography. Losses of renatured material could be reduced, or higher protein concentrations used during refolding, if the denatured product was purified by cation-exchange chromatography in urea prior to refolding. Apart from an additional N-terminal N-acetyl methionine, the refolded product proved identical to human serum albumin derived from plasma when compared by a variety of physical, chemical, and biological analytical methods.