Prokaryotic Expression of the Oligopeptide Transporter hPepT2(560-663)
School of Chemistry and Chemical Engineering, Henan University of Technology, Zhengzhou 450001 1Chemical Research Institute Co., Ltd., of Henan Province, Zhengzhou 450052 2School of Material and Chemical Engineering, Henan Institute of Engineering, Zhengzhou 451191, China
K. Lu School of Material and Chemical Engineering, Henan Institute of Engineering, Zhengzhou 451191, China E-mail: [email protected]
Peptide transporter 2 is a high affinity and low capacity transporter, that has recently been found in the mammalian kidney, brain, lung, and mammary glands, which is known to transport dipeptides and tripeptides and to reabsorb small peptides generated by luminal peptidases in the kidney. In addition, peptide transporter 2 can transport peptide-like drugs and play important roles in the transshipment and positioning of drugs. In this paper, an E.coli BL21(DE3) containing pET30a(+)-hPepT2(560-663) recombinant plasmid was obtained by using PCR, double restriction enzyme digestion, ligation, and transformation. Then, the effect of changing both the induction time and induction dose using Isopropyl β-D-1-Thiogalactopyranoside (IPTG) on the bacterial expression of hPepT2(560-663) was detected by SDS-PAGE electrophoresis, the optimum expression conditions (at 37°) were identified as: 3 h and a working concentration of 0.5 mM Isopropyl β-D-1-Thiogalactopyranoside. Finally, we purified the target protein hPepT2(560-663) via Sephadex G-75 gel chromatography. These results will facilitate in future studies on the nature and function of the protein.