Modulation of chaperone gene expression in mutagenized Saccharomyces cerevisiae strains developed for recombinant human albumin production results in increased production of multiple heterologous proteins.

The yeast Saccharomyces cerevisiae has been successfully established as a commercially viable system for the production of recombinant proteins. Manipulation of chaperone gene expression has been utilized extensively to increase recombinant protein production from S. cerevisiae, focusing predominantly on the products of the protein disulfide isomerase gene PDI1 and the hsp70 gene KAR2. Here we show that the expression of the genes SIL1, LHS1, JEM1, and SCJ1, all of which are involved in regulating the ATPase cycle of Kar2p, is increased in a proprietary yeast strain, developed by several rounds of random mutagenesis and screening for increased production of recombinant human albumin (rHA). To establish whether this expression contributes to the enhanced-production phenotype, these genes were overexpressed both individually and in combination. The resultant strains showed significantly increased shake-flask production levels of rHA, granulocyte-macrophage colony-stimulating factor, and recombinant human transferrin.

Yeast 2 microm plasmid copy number is elevated by a mutation in the nuclear gene UBC4.

The copy number of the Saccharomyces cerevisiae endogenous 2 microm plasmid is under strict control to ensure efficient propagation to the daughter cell without significantly reducing the growth rate of the mother or the daughter cell. A recessive mutation has been identified that resulted in an elevated but stable 2 microm plasmid copy number, which could be complemented by a genomic DNA clone containing the UBC4 gene, encoding an E2 ubiquitin-conjugating enzyme. A ubc4::URA3 deletion resulted in the same elevated 2 microm plasmid copy number. An analysis of the endogenous 2 microm transcripts revealed that the steady-state abundance of REP1, REP2, FLP and RAF were all increased 4-5-fold in the mutant. Analysis of the mutant ubc4 allele identified a single base pair mutation within the UBC4 coding region, which would generate a glutamic acid to lysine amino acid substitution within a region of conserved tertiary structure located within the first alpha-helix of Ubc4p. These investigations represent the first molecular characterization of a mutation within a Saccharomyces cerevisiae nuclear gene shown to affect 2 microm steady-state plasmid copy number and implicate the ubiquitin-dependent proteolytic pathway in host control of 2 microm plasmid copy number.

Crystallization and X-ray diffraction study of recombinant platelet-derived endothelial cell growth factor.

Crystals of recombinant platelet-derived endothelial cell growth factor (PD-ECGF) were obtained by the hanging drop vapour diffusion technique. The crystals belong to the space group P2(1)2(1)2(1) with unit cell dimensions a = 63.7 A, b = 70.4 A, c = 219.6, alpha = beta = gamma = 90 degrees, and probably contain a single dimer in the asymmetric unit. Diffraction to a minimum Bragg spacing of 3.5 A has been obtained using a synchrotron X-ray source.

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 recombinant platelet-derived endothelial cell growth factor in the yeast Saccharomyces cerevisiae.

A platelet-derived endothelial cell growth factor cDNA has been cloned, sequenced and expressed using the Saccharomyces cerevisiae PRB1 promoter. Soluble recombinant platelet-derived endothelial cell growth factor constituted 0.5-1.0% of total soluble protein. Yeast soluble protein extracts containing recombinant platelet-derived endothelial cell growth factor stimulate the growth of calf pulmonary artery endothelial cells in vitro.