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 alpha 3/beta 1 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 alpha 3/beta 1 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 alpha 3- and beta 1-subunits and even coexpress them after methanol induction. beta 1-subunit was produced as a major 44-kDa glycosylated polypeptide and alpha 3 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.
|Titolo:||Expression of the alpha3/beta1 isoform of human Na,K-ATPase in the methylotrophic yeast Pichia pastoris|
POPOLO, LAURA MARIA (Ultimo)
|Parole Chiave:||Pichia pastoris; Na/K pump; Na,K-ATPase alpha 3-subunit; Na,K-ATPase beta 1-subuni; protein expressio; protein glycosylation|
|Settore Scientifico Disciplinare:||Settore BIO/11 - Biologia Molecolare|
|Data di pubblicazione:||2007|
|Digital Object Identifier (DOI):||10.1111/j.1567-1364.2007.00227.x|
|Appare nelle tipologie:||01 - Articolo su periodico|