Recovery of functional peptide transporter PepTl in budded baculovirus fraction

• Published in: Protein expression and purification 2006-03, Vol.46 (1), p.130-135
• Main Authors: Saitoh, R, Ohtomo, T, Ito, Y, Nezu, J-I, Kimura, N, Funahashi, S-I, Aso, Y, Ohizumi, I, Kodama, T, Hamakubo, T, Tsuchiya, M
• Format: Article
• Language: English
• Subjects: Baculovirus
• ISSN: 1046-5928
• DOI: 10.1016/j.pep.2005.08.008

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 [ super(14)C]glycylsarcosine was markedly increased in an acidic condition and showed a clear overshoot in PepT1-expressing virus fraction. The apparent Michaelis constant for [ super(14)C]glycylsarcosine was 0.55 plus or minus 0.06 mM. [ super(14)C] 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.