Expression of the myc/His-tagged human peptide transporter hPEPT1 in yeast for protein purification and functional analysis.
The human intestinal peptide transporter hPEPT1 has been expressed in the yeast Pichia pastoris using the promoter of the glyceraldehyde-3-phosphate-dehydrogenase gene. A myc-epitope fused to a polyhistidine-tag was introduced at the C-terminus of hPEPT1 for ease of detection and purification. Yeast cells transformed with tagged hPEPT1 exhibited 30-fold increased dipeptide uptake compared to control cells with a substrate specificity and pH dependence similar to the native transporter. The tagged hPEPT1 protein was detected in crude membrane fractions of Pichia cells with an apparent molecular mass of 66 kDa and an expression level of approximately 64 pmol/mg membrane protein. These studies demonstrate that tagged hPEPT1 can be expressed functionally in P. pastoris with unaltered phenotypical characteristics allowing the yeast cells to be used for functional analysis such as screening for compounds utilizing the peptide transporter for absorption in the human intestine. Moreover, recombinant hPEPT1 can now easily be detected for further purification purposes using immobilized metal-affinity chromatography.
Theis
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S
Molecular Nutrition Unit, Institute of Nutritional Sciences, Technical University of Munich, Hochfeldweg 2, D-85350 Freising-Weihenstephan, Germany.
Döring
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F
Daniel
H
H
D000900:Anti-Bacterial Agents / Q000737:chemistry; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D016259:Genes, myc; D006639:Histidine; D006801:Humans; D007769:Lactams; D000074011:Peptide Transporter 1; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis; D027981:Symporters
D000900:Anti-Bacterial Agents; D002352:Carrier Proteins; D007769:Lactams; D000074011:Peptide Transporter 1; D011994:Recombinant Proteins; C082186:SLC15A1 protein, human; D027981:Symporters; D006639:Histidine