Expression of polyoma virus middle-T antigen in Saccharomyces cerevisiae

Abstract

The polyoma middle-T gene, lacking its intron, was inserted into a yeast expression plasmid containing the phosphoglycerate kinase promoter. Such plasmids transformed yeast at low frequency and these transformants expressed middle-T antigen at a level of approximately 0.1 % cell protein. Furthermore, expression of this protein was frequently lost during growth in liquid culture and this loss of middle-T was accompanied by a twofold increase in the rate of growth. The spontaneous production of a truncated middle-T antigen, lacking the C terminus, was also observed; the expression of this protein did not inhibit the growth rate of the cells. Recovery and analysis of the expression plasmids encoding the truncated molecule showed that a single C . G base pair had been deleted from a run of nine consecutive C . G base pairs (Pyr nucleotide 1239 -1247) within the middle-T coding region. This frame-shift mutation results in premature termination of the protein and loss of the strongly hydrophobic region of the molecule believed to be responsible for the membrane association of middle-T antigen.

Expression in High Yield of Pig α1β1 Na,K-ATPase and Inactive Mutants D369N and D807N in Saccharomyces cerevisiae

“…Previous experiments with membrane proteins, OmpA (49) and ␣-hemagglutinin (50), demonstrated selection against the production of hydrophobic intramembrane segments in response to the poor tolerance by the host cell. Selection for low expressing variants has been observed in several cases, where constitutive promoters were used to drive expression of membrane proteins (51,52). In the present expression system, the selection pressure was avoided through the use of a strong inducible promoter, but the toxicity of the expression suggests that the capacity of the yeast membrane system may set the limit for the concentration of Na,K-ATPase that can be achieved in these cells.…”

Alteration in membrane lipid order and composition in metabolically hyperactive fatty rat adipocytes

“…Cells were homogenized in a buffer containing 10 mM tris(hydroxymethyl)aminomethane (Tris), 1 mM ethylenediaminetetraacetic acid and 255 mM sucrose, pH 7.4, at 4~ and the homogenates were centrifuged at 16,000 x g max for 15 min. Plasma membrane fractions were prepared from the 16,000 x g pellet by centrifugation on a PercolYsucrose gradient (15). High density microsomes (HDM) and low density microsomes (LDM) were prepared by differential ultracentrifugation of the 16,000 x g supernatant, as described previously (5).…”

Factors affecting homologus overexpression of the Sacchromyces cerevisiae Lanosterol 14α‐demethylase gene

“…Regulated gene expression is indispensable if gene products causing highly toxic effects upon yeast cells are to be produced (Meyhack et al, 1989;Barnes and Rine, 1985;Pines et al, 1988). Even if the heterologous protein to be produced is not directly lethal for the host cells, the regulated situation may be more desirable in terms of yield (Weber et al, 1990;Bitter and Egan, 1988;Legrain et al, 1989), quality of the gene product (Belsham et al, 1986), plasmid stability (Meyhack et al, 1989;Bitter and Egan, 1988;Jabbar et al, 1985), growth behaviour (Meyhack et al, 1989;Bitter and Egan, 1988;Mak et al, 1989) and bioenergetic consequences (Gopal et al, 1989). Since 14DM overproduction leads to a competition for available heme molecules in the cells, 14DM gene overexpression could, therefore, change the metabolic state of the cells, resulting in a reduced growth rate and/or a decrease in plasmid copies in the case of constitutive expression.…”