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Titolo:
EXPRESSION OF THE MAMMALIAN RENAL PEPTIDE TRANSPORTER PEPT2 IN THE YEAST PICHIA-PASTORIS AND APPLICATIONS OF THE YEAST SYSTEM FOR FUNCTIONAL-ANALYSIS
Autore:
DORING F; MICHEL T; ROSEL A; NICKOLAUS M; DANIEL H;
Indirizzi:
UNIV GIESSEN,INST NUTR SCI,WILHELMSTR 20 D-35392 GIESSEN GERMANY UNIV GIESSEN,INST NUTR SCI D-35392 GIESSEN GERMANY
Titolo Testata:
Molecular membrane biology
fascicolo: 2, volume: 15, anno: 1998,
pagine: 79 - 88
SICI:
0968-7688(1998)15:2<79:EOTMRP>2.0.ZU;2-X
Fonte:
ISI
Lingua:
ENG
Soggetto:
HIGH-LEVEL SECRETION; METHYLOTROPHIC YEAST; GENE; OLIGOSACCHARIDES; INVERTASE; STRAINS; DOMAIN;
Keywords:
RENAL PEPTIDE TRANSPORTER; HETEROLOGOUS EXPRESSION; PICHIA PASTORIS; FLUORESCENT DIPEPTIDE ANALOG;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Science Citation Index Expanded
Citazioni:
26
Recensione:
Indirizzi per estratti:
Citazione:
F. Doring et al., "EXPRESSION OF THE MAMMALIAN RENAL PEPTIDE TRANSPORTER PEPT2 IN THE YEAST PICHIA-PASTORIS AND APPLICATIONS OF THE YEAST SYSTEM FOR FUNCTIONAL-ANALYSIS", Molecular membrane biology, 15(2), 1998, pp. 79-88

Abstract

It has recently been identified that PEPT2 cDNA encodes the high affinity proton-coupled peptide transporter in rabbit kidney cortex. PEPT2represents a 729 amino acid protein with 12 putative transmembrane domains that mediates H+/H3O+ dependent electrogenic transmembrane transport of di- and tripeptides and of selected peptidomimetics. Here the functional expression of PEPT2 in the methylotropic yeast Pichia pastoris is described under the control of a methanol inducible promoter. Western blot analysis of Pichia cell membranes prepared from a recombinant clone identified a protein with an apparent molecular mass of about 85-87 kDa. Peptide uptake into cells expressing PEPT2 was up to 80 times higher than in control cells. Cells of recombinant clones showed a saturable peptide transport activity for the hydrolysis resistant dipeptide H-3-D-Phe-Ala with an app. K-0.5 Of 0.143 +/- 0.016 mM. Inhibition of H-3-D-PheAla uptake by selected di- and tripeptides and beta-lactam antibiotics revealed the same substrate specificity as obtained In renal membrane vesicles or for PEPT2 when expressed in Xenopus laevis oocytes. A novel fluorescence based assay for assessing transport function based on a coumarin-labeled fluorescent peptide analogue has also been developed. Moreover, using a histidyl auxotrophe strain a PEPT2 expressing cell clone in which transport function can be monitored by a simple yeast growth test was established. In conclusion, this is one of only a few reports on successful functional expression of mammalian membrane transport proteins in yeast. The high expression level will provide a simple means for future studies either on the structure-affinity relationship for substrate interaction with PEPT2 or for selection of mutants generated by random mutagenesis.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 31/05/20 alle ore 11:41:46