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Expression and Functional Characterization of the Mammalian Intestinal Peptide Transporter PepT1 in the Methylotropic YeastPichia pastoris,☆☆

https://doi.org/10.1006/bbrc.1997.6351Get rights and content

Abstract

The methylotrophic yeastPichia pastoriswas 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 β-lactam antibiotics and ACEinhibitors. Functional expression of PepT1 was determined in different recombinant clones by flux studies employing the radiolabeled dipeptide3H-(D)-Phe-(L)-Ala. One clone (GS-PepT1) displayed high level functional expression that was pH dependent and saturable with an app. K0.5of 1.17 ± 0.18 mM. Inhibition of3H-(D)-Phe-(L)-Ala uptake into GS-PepT1 by selected dipeptides, tripeptides and peptidomimetics including β-lactam antibiotics and ACE-inhibitors revealed the same substrate specifity as reported for PepT1 when expressed in mammalian cells orXenopus laevisoocytes.Pichiacells 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 usingP. pastoris.

References (25)

  • J.J. Clare et al.

    Gene

    (1991)
  • K.I. Miyamoto et al.

    BBA

    (1996)
  • R. Liang et al.

    J. Biol. Chem.

    (1995)
  • T. Terada et al.

    FEBS Letters

    (1996)
  • B. Mackenzie et al.

    J. Biol. Chem.

    (1996)
  • F. Naider et al.

    J. Biol. Chem.

    (1974)
  • J. Slavik et al.

    BBA

    (1984)
  • J.M. Cregg et al.

    Bio/Technology

    (1993)
  • R. Couderc et al.

    Agric. Biol. Chem.

    (1980)
  • J.J. Clare et al.

    Bio/Technology

    (1991)
  • Y. Laroche et al.

    Bio/Technology

    (1994)
  • R. Ridder et al.

    Bio/Technology

    (1995)
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    This work was supported by the Deutsche Forschungsgemeinschaft, Projekt B12, SFB 249 to H. Daniel.

    ☆☆

    Abbreviations: PepT1, rabbit intestinal peptide transporterAOX1, alcohol oxidase1; MutS, slow methanol utilization; Mut+, same methanol utilization pathway as wild type; ACE, angiotensin converting enzyme; OD, optical density 600nm (one OD equals approximately 5×107cells);

    2

    Corresponding author. Institute of Nutritional Sciences, Justus-Liebig-University Giessen, Wilhelmstrasse 20, D-35392 Giessen, Germany. Fax: Germany 641 99 39049. E-mail: hannelore.daniel@ ernaehrung.uni-giessen.de.

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