Expression of the α3/β1 isoform of human Na,K-ATPase in the methylotrophic yeast Pichia pastoris

Abstract

Na,K-ATPase is a crucial enzyme for ion homeostasis in human tissues. Different isozymes are produced by assembly of four α- and three β-subunits. The expression of the α3/β1 isozyme is confined to brain and heart. Its heterologous production has so far never been attempted in a lower eukaryote. In this work we explored whether the methylotrophic yeast Pichia pastoris is capable of expressing the α3/β1 isoform of human Na,K-ATPase. cDNAs encoding the α3 and the β1- subunits were cloned under the control of the inducible promoter of Pichia pastoris alcohol oxidase 1. Pichia pastoris could express the single α3- and β1-subunits and even coexpress them after methanol induction. β1-subunit was produced as a major 44-kDa glycosylated polypeptide and α3 as a 110-kDa unglycosylated polypeptide. Expression at the plasma membrane was limited in shaking flask cultures but by cultivating P. pastoris cells in a fermenter there was a 10-fold increase of the number of ouabain binding sites per cell. The exported enzyme was estimated to be about 0.230 mg L-1 at the end of a bioreactor run. Na,K-ATPase proved active and the dissociation constant of the recombinant enzyme-ouabain interaction was determined. © 2007 Federation of European Microbiological Societies.