EXPRESSION IN HIGH-YIELD OF PIG ALPHA-1-BETA-1 NA,K-ATPASE AND INACTIVE MUTANTS D369N AND D807N IN SACCHAROMYCES-CEREVISIAE

Studies of structure-function relationships in Na,K-ATPase require hig h yield expression of inactive mutations in cells without endogenous N a,K-ATPase activity. In this work we developed a host/vector system fo r expression of fully active pig Na,K-ATPase as well as the inactive m utations D369N and D807N at high levels in Saccharomyces cerevisiae. T he alpha 1- and beta 1-subunit cDNAs were inserted into a single 2-mu m-based plasmid with a high and regulatable copy number and strong gal actose inducible promoters allowing for stoiehiometric alterations of gene dosage. The protease-deficient host strain was engineered to expr ess high levels of GAL4 transactivating protein, thereby causing a 10- fold increase in expression to 32,500 +/- 3,000 [H-3]ouabain sites/cel l. In one bioreactor run 150-200 g of yeast were produced with 54 +/- 5 mu g of Na,K-pump protein/g of cells. Through purification in membra ne bound form the activity of the recombinant Na,K-ATPase was increase d to 42-50 pmol/mg of protein. The Na,K dependence of ATP hydrolysis a nd the molar activity (4,500-7,000 min(-1)) were close to those of nat ive pig kidney Na,K-ATPase. Mutations to the phosphorylation site (D36 9N) or presumptive cation sites (D807N), both devoid of Na,K-ATPase ac tivity, were expressed in the yeast membrane at the same alpha-subunit concentration and [H-3]ouabain binding capacity as the wild type Na,K -ATPase. The high yield and absence of endogenous activity allowed ass ay of [H-3]ATP binding at equilibrium, demonstrating a remarkable 18-f old increase in affinity for ATP in consequence of reducing the negati ve charge at the phosphorylation site (D369N).