Studies of structure-function relationship in Na.K ATPasr require high yield expression of inactive mutants in cells without endogenous Na,K-ATPase activity. We have developed a host/vector system for expression of fully active pig kidney la,K-ATPase as well as inactive mutants specifically engaged in nucleotide or cation interactions. The ol and /M-subunit cDNAs were inserted into a single 2μ based plasrnid with a high and regulatable copy number and strong galactose inducible promoters allowing for stoic.hiometiic alterations of gene dosage and induction. The protease deficient host strain was engineered to express high levels of GAI.4 trans activating piotein thereby causing a 10 fold increase in expression to 32,500 n 3000 [³H]-ouabain binding sites per cell. In one bioreactor run 150-200 g yeast were produced with >1 n 5 //g iS'a,K-pump protein per g cells. A series of mutations was constructed in a very conserved stretch of amino acid residues potentially engaged in AT P binding (residues 712 722 in pig kidney al-subunit). These mutations could not be expressed in our usual expression system due to an increased rate of degradation. We managed to prevent these mutants from being degraded by alteration of growth conditions, addition of protease inhibitors prior to induction and construction of a new yeast host cell. This work was supported by the Danish Research Council, the Novo-Nordic Foundation and the Carlsberg Foundation.