Döring F, Theis S, Daniel H
Institute of Nutritional Sciences, University of Giessen, Germany.
Biochem Biophys Res Commun. 1997 Mar 27;232(3):656-62. doi: 10.1006/bbrc.1997.6351.
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.
甲基营养型酵母巴斯德毕赤酵母被用于兔肠道肽转运体PepT1的异源表达及其功能表征。PepT1介导二肽、三肽以及肽模拟物（如β-内酰胺抗生素和血管紧张素转换酶抑制剂）的电驱动跨膜转运。通过使用放射性标记的二肽3H-(D)-Phe-(L)-Ala的通量研究，在不同的重组克隆中测定了PepT1的功能表达。一个克隆（GS-PepT1）表现出高水平的功能表达，该表达依赖于pH值且具有饱和性，其表观K0.6为1.17±0.18 mM。包括β-内酰胺抗生素和血管紧张素转换酶抑制剂在内的选定二肽、三肽和肽模拟物对GS-PepT1摄取3H-(D)-Phe-(L)-Ala的抑制作用，揭示了与在哺乳动物细胞或非洲爪蟾卵母细胞中表达时报道的PepT1相同的底物特异性。表达PepT1的毕赤酵母细胞将为肽和肽模拟物的体外生物利用度研究提供一个出色的工具。此外，据我们所知，这是首次使用巴斯德毕赤酵母对哺乳动物膜转运蛋白进行功能表达的证明。
