title: Recovery of functional peptide transporter PepT1 in budded baculovirus fraction.
abstract: Transporters play a critical role in many physiological and pathological states and expression of the functional transporter protein is essential in exploring its kinetics and developing effective drugs. We describe here the recovery of functional transporter protein in the baculovirus fraction. We introduced a gene encoding human peptide transporter PepT1, important for the absorption of protein hydrolytic products or peptide-mimetic drugs, into a baculovirus vector. After infection, a large amount of PepT1 appeared in the budded virus fraction compared with Sf9 cells. Uptake of [14C]glycylsarcosine was markedly increased in an acidic condition and showed a clear overshoot in PepT1-expressing virus fraction. The apparent Michaelis constant for [14C]glycylsarcosine was 0.55 +/- 0.06 mM. [14C]Glycylsarcosine uptake was inhibited by di- and tripeptides and orally active beta-lactam antibiotics. These results suggest that functional PepT1 recovers efficiently in a budded virus fraction, and, thus, this expression system will be a useful tool for characterization and screening of peptide-mimetic drugs in drug discovery.
authors.affiliation: Fuji Gotemba Research Laboratories, Chugai Pharmaceutical Co. Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan. saitorui@chugai-pharm.co.jp
authors.forename: Ryoichi
authors.initials: R
authors.lastname: Saitoh
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Ohtomo
authors.forename: Yukio
authors.initials: Y
authors.lastname: Ito
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Nezu
authors.forename: Naoki
authors.initials: N
authors.lastname: Kimura
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Funahashi
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Aso
authors.forename: Iwao
authors.initials: I
authors.lastname: Ohizumi
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Kodama
authors.forename: Takao
authors.initials: T
authors.lastname: Hamakubo
authors.forename: Masayuki
authors.initials: M
authors.lastname: Tsuchiya
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular; D006801:Humans; D007700:Kinetics; D008854:Microscopy, Electron; D000074011:Peptide Transporter 1; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification; D015183:Restriction Mapping; D018411:Spodoptera; D027981:Symporters / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D000074011:Peptide Transporter 1; D011994:Recombinant Proteins; C082186:SLC15A1 protein, human; D027981:Symporters