Expression of the myc/His-tagged human peptide transporter hPEPT1 in yeastfor protein purification and functional analysis

The human intestinal peptide transporter hPEPT1 has been expressed in the y east Pichia pastoris using the promoter of the glyceraldehyde-3-phosphate-d ehydrogenase gene. A myc-epitope fused to a polyhistidine-tag was introduce d 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 depen dence similar to the native transporter. The tagged hPEPT1 protein was dete cted in crude membrane fractions of Pichia cells with an apparent molecular mass of 66 kDa and an expression level of approximately 64 pmol/mg membran e protein. These studies demonstrate that tagged hPEPT1 can be expressed fu nctionally in P. pastoris with unaltered phenotypical characteristics allow ing the yeast cells to be used for functional analysis such as screening fo r compounds utilizing the peptide transporter for absorption in the human i ntestine. Moreover, recombinant hPEPT1 can now easily be detected for furth er purification purposes using immobilized metal-affinity chromatography. ( C) 2001 Academic Press.