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 high yield expression of inactive mutations in cells without endogenous Na,K-ATPase activity. In this work we developed a host/vector system for expression of fully active pig Na,K-ATPase as well as the inactive mutations D369N and D807N at high levels in Saccharomyces cerevisiae. The alpha 1- and beta 1-subunit cDNAs were inserted into a single 2-microns-based plasmid with a high and regulatable copy number and strong galactose-inducible promoters allowing for stoichiometric alterations of gene dosage. The protease-deficient host strain was engineered to express high levels of GAL4 transactivating protein, thereby causing a 10-fold increase in expression to 32,500 +/- 3,000 [3H]ouabain sites/cell. In one bioreactor run 150-200 g of yeast were produced with 54 +/- 5 micrograms of Na,K-pump protein/g of cells. Through purification in membrane bound form the activity of the recombinant Na,K-ATPase was increased to 42-50 pmol/mg of protein. The Na,K dependence of ATP hydrolysis and the molar activity (4,500-7,000 min-1) were close to those of native pig kidney Na,K-ATPase. Mutations to the phosphorylation site (D369N) or presumptive cation sites (D807N), both devoid of Na,K-ATPase activity, were expressed in the yeast membrane at the same alpha-subunit concentration and [3H]ouabain binding capacity as the wild type Na,K-ATPase. The high yield and absence of endogenous activity allowed assay of [3H]ATP binding at equilibrium, demonstrating a remarkable 18-fold increase in affinity for ATP in consequence of reducing the negative charge at the phosphorylation site (D369N).
Pedersen
P A
PA
Biomembrane Research Center, August Krogh Institute, University of Copenhagen, Denmark.
Rasmussen
J H
JH
Jøorgensen
P L
PL
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D017931:DNA Primers; D005822:Genetic Vectors; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013552:Swine
D017931:DNA Primers; D011994:Recombinant Proteins; D001224:Aspartic Acid; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase