Expression and functional characterization of the mammalian intestinal peptide transporter PepT1 in the methylotropic yeast Pichia pastoris.
The methylotrophic yeast Pichia pastoris was used for heterologous expression of the rabbit intestinal peptide transporter PepT1 and its functional characterization. PepT1 mediates the electrogenic transmembrane transport of di- and tripeptides and peptido-mimetics such as beta-lactam antibiotics and ACE-inhibitors. Functional expression of PepT1 was determined in different recombinant clones by flux studies employing the radiolabeled dipeptide 3H-(D)-Phe-(L)-Ala. One clone (GS-PepT1) displayed high level functional expression that was pH dependent and saturable with an app. K0.6 of 1.17 +/- 0.18 mM. Inhibition of 3H-(D)-Phe-(L)-Ala uptake into GS-PepT1 by selected dipeptides, tripeptides and peptidomimetics including beta-lactam antibiotics and ACE-inhibitors revealed the same substrate specifity as reported for PepT1 when expressed in mammalian cells or Xenopus laevis oocytes. Pichia cells expressing PepT1 will provide an excellent tool for in vitro bioavailability studies for peptides and peptidomimetics. Moreover, to our knowledge, this is the first demonstration of functional expression of a mammalian membrane transport protein using P. pastoris.
Döring
F
F
Institute of Nutritional Sciences, University of Giessen, Germany.
Theis
S
S
Daniel
H
H
D000818:Animals; D001483:Base Sequence; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D003470:Culture Media; D018076:DNA, Complementary / Q000235:genetics; D004151:Dipeptides / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D000074011:Peptide Transporter 1; D010455:Peptides / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D027981:Symporters
D002352:Carrier Proteins; D003470:Culture Media; D018076:DNA, Complementary; D004151:Dipeptides; D000074011:Peptide Transporter 1; D010455:Peptides; D011994:Recombinant Proteins; D027981:Symporters; C039552:phenylalanylalanine