Expression of the alpha3/beta1 isoform of human Na,K-ATPase in the methylotrophic yeast Pichia pastoris.
Na,K-ATPase is a crucial enzyme for ion homeostasis in human tissues. Different isozymes are produced by assembly of four alpha- and three beta-subunits. The expression of the alpha3/beta1 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 alpha3/beta1 isoform of human Na,K-ATPase. cDNAs encoding the alpha(3) and the beta(1)-subunits were cloned under the control of the inducible promoter of Pichia pastoris alcohol oxidase 1. Pichia pastoris could express the single alpha3- and beta1-subunits and even coexpress them after methanol induction. beta1-subunit was produced as a major 44-kDa glycosylated polypeptide and alpha3 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.
Reina
Cristina
C
Prassis Sigma-Tau Research Institute, Settimo Milanese, Milan, Italy.
Padoani
Gloria
G
Carotti
Cristina
C
Merico
Annamaria
A
Tripodi
Grazia
G
Ferrari
Patrizia
P
Popolo
Laura
L
D019149:Bioreactors; D018076:DNA, Complementary; D015870:Gene Expression; D006801:Humans; D007527:Isoenzymes / Q000235:genetics; D010843:Pichia / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics*
D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D000254:Sodium-Potassium-Exchanging ATPase