Each species cDNA was digested with the appropriate restriction enzymes (BamHI/AvrII for human and rat and BglII/XbaI for mouse), ligated into the plasmid DNA vector pPIC3.5K, and then transformed into DH5 competent cells. cells expressing these mammalian species. Materials and Methods Materials. [3H]Glycylsarcosine (98 mCi/mmol) and [14C]polyethylene glycol (PEG) 200 (1.1 mCi/g) were purchased from Moravek Biochemicals (Brea, CA). [14C]Oseltamivir phosphate (103 Ci/mg) and unlabeled oseltamivir phosphate were gifts of F. Hoffmann-La Roche (Basel, Switzerland). Biotin, AccuTaq LA DNA Polymerase, and unlabeled glycylsarcosine (GlySar) was purchased from Sigma-Aldrich (St. Louis, MO). SuperScript III reverse transcriptase, DH5 qualified cells, GS115 strain, and vector pPIC3.5K were purchased from Invitrogen (Carlsbad, CA). The human Pept1 cDNA was a gift from Matthias Hediger (University or college of Bern, Bern, Switzerland). All other chemicals were acquired from standard sources. PCR Amplification of Pept1 cDNA and Construction of Expression Vector pPIC3.5K-Pept1. Rat, mouse, and human Pept1 cDNA were cloned by proofreading PCR with species-specific primers (Table 1) using the reverse transcripts of rat or mouse small intestine total RNA or from a vector made up of the human cDNA. Each species cDNA was digested with the appropriate restriction enzymes (BamHI/AvrII for human and rat and BglII/XbaI for mouse), ligated into the plasmid DNA vector pPIC3.5K, and then transformed into DH5 competent cells. Positive colonies were screening by PCR with a pair of primers designed for an internal fragment of the Pept1 gene (Table 2). Once positive colonies were obtained, plasmid DNA was purified using the PureYield Plasmid Midiprep System (Promega, Madison, WI) GSK2801 after which each species-specific plasmid DNA was sequenced on both strands of the entire Pept1 gene by the DNA Sequencing Core, University or college of Michigan. TABLE 1 Primers for Pept1 cDNA cloning GS115. The procedure was performed as explained Mouse Monoclonal to MBP tag in Manual Version M of the Pichia Expression Kit (Invitrogen). In brief, plasmid DNA made up of each species of Pept1 cDNA was linearized by the restriction enzyme SalI and then purified with the QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA). A 10-ng aliquot of linearized plasmid DNA was used to transform GS115 by electropermeabilization using a MicroPulser Electroporator (Bio-Rad Laboratories, Hercules, CA). The GS115 yeast cells were then cultured on MM (1.34% YNB, 4 10?5% biotin, 0.5% methanol, and 1.5% agarose) and MD (1.34% YNB, 4 10?5% biotin, 2% dextrose, 1.5% agarose) plates for distinguishing the His+Mut+ from His+Muts transformants. After isolation of genomic DNA from your His+Mut+ GS115 transformants, real-time PCR was performed with species-specific primers (Table 3) to measure the gene copy quantity of Pept1 cDNA in yeast cells (Abad et al., 2010). The gene of yeast was set as the internal control, and the plasmid DNA pPIC3.5K/Pept1 was set as the positive control. TABLE 3 Primers for measuring Pept1 integration Strains and PEPT1 Protein. The procedure was performed as explained in Manual Version M of the Pichia Expression Kit. In brief, His+Mut+ transformants made up of pPIC3.5K (vector alone), pPIC3.5K-hPept1 (human), pPIC3.5K-mPept1 (mouse), and pPIC3.5K-rPept1 (rat) were inoculated on MM and MD plates and incubated at 30C for 2 days. A single colony of each specific plasmid was picked from your MD plate, transferred into a 100-ml baffled flask made up of 10 ml of BMGY medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, GSK2801 1.34% YNB, 4 10?5% biotin, and 1% glycerol) and produced overnight in a shaking incubator at 30C. After centrifugation at 3000for 5 min at room heat, the cell pellet was suspended in 100 ml of BMMY media (1% yeast extract, 2% peptone, 100 mM GSK2801 potassium phosphate, pH 6.0, 1.34% YNB, 4 10?5% biotin, and 0.5% methanol) and produced overnight in a shaking baffled flask at 30C for inducing PEPT1 protein expression. Transport Assay in Yeast. Uptake studies were performed with radiolabeled GlySar or oseltamivir (after a 20- to 24-h induction of PEPT1 expression) using a method explained previously (D?ring et al., 1997, 1998). Cell cultures were harvested by centrifugation at 3000for 5 min at room temperature, washed with the same volume of 100 mM potassium phosphate buffer, pH 6.5 (PPB), centrifuged, and resuspended in one-half the original volume of 100 mM PPB, centrifuged, resuspended in one-tenth the volume of 100 mM PPB, and stored on ice. All uptake measurements were performed at 24C unless normally indicated. Uptake was initiated by rapidly combining 20 l of yeast cell suspension and 30 l of PPB GSK2801 made up of 0.05 Ci GSK2801 of [3H]GlySar (final concentration of 5.0 M) and then incubating for the designated time period. For concentration-dependent studies (0.005C10 mM GlySar), the reaction was terminated at 30 s, a time shown in preliminary experiments to reflect linear uptake kinetics. For specificity studies, [3H]GlySar (5.0 M) was incubated for 30 s in the presence of potential inhibitors (10 mM) such as amino acids (glycine and l-histidine), a dipeptide [glyclyproline (GlyPro)], cephalosporins with (cefadroxil and cephradine) and without (cefazolin, cephalothin, and.