Recovery of functional peptide transporter PepT1 in budded baculovirus fraction.
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.
Saitoh
Ryoichi
R
Fuji Gotemba Research Laboratories, Chugai Pharmaceutical Co. Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan. saitorui@chugai-pharm.co.jp
Ohtomo
Toshihiko
T
Ito
Yukio
Y
Nezu
Jun-ichi
J
Kimura
Naoki
N
Funahashi
Shin-ichi
S
Aso
Yoshinori
Y
Ohizumi
Iwao
I
Kodama
Tatsuhiko
T
Hamakubo
Takao
T
Tsuchiya
Masayuki
M
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
D000074011:Peptide Transporter 1; D011994:Recombinant Proteins; C082186:SLC15A1 protein, human; D027981:Symporters