Prokaryotic Expression And Properties Of Oligopeptide Transporter HPepT2(560-664)

PEPT1(SLC15A1) and PEPT2(SLC15A2) belong to the solute carrier (SLC) family ofthe proton-dependent oligopeptide transporters, along with the peptide histidine transportersPHT1(SLC15A4) and PHT2(SLC15A3). PEPT2and PEPT1peptide transporters arepolytopic integral membrane proteins with12membrane-spanning domains with their N-andC-terminus facing the cytosol. The primary structures of the proteins exhibit50%identity and70%similarity. PEPT2is expressed in a variety of organs including kidney, lung, brain,mammary gland, pituitary gland, testis, prostate, ovary, uterus, eye and the mammalianenteric nervous system. PEPT2is a high-affinity and low-capacity transporter. PEPT2cantransport numerous peptides including400different dipeptides and8000different tripeptides,and covering a wide range of molecular weights from96.2Da(di-Gly) to522.6Da(tri-Trp),and also transport peptidomimetics such as β-lactam antibiotics, penicillin antibiotics,cephalosporins, glycyl sarcosine, anti-cancer drug phenylbutyrate inhibin, renin inhibitors,angiotensin I converting enzyme (ACE) inhibitors and thrombin inhibitors. It is generallyassumed that PEPT2displays a10–20times higher affinity to its substrates than PEPT1.Because PEPT2is widely distributed in the human body, it is responsible for thereabsorption of di-and tri-peptides. Moreover, PEPT2has a significant influence on the invivo disposition and half-life time of peptide-like drugs within the body, which might play amajor role in future treatment of various pulmonary and systemic diseases. Therefore thepeptide transporter hPEPT2was researched in this study. First, the E.coli BL21(DE3)containing pET30a(+)-hPEPT2(560-664) recombinant plasmid was obtained after PCR,double restriction enzyme digestion, connection and transformation. Then, the effect ofchanging the induction time and induction dose to the expression of hPEPT2(560-664) wasdetected by SDS-PAGE electrophoresis. It was found that the optimum expression conditionswere at37°C for3h and the working concentration of IPTG was0.5mM. The target proteinhPEPT2(560-664) was purified through Sephadex G-75gel chromatography. We researchedthe nature of the hPEPT2(560-664) in different pH buffers by UV and fluorescence spectroscopy and analysed the secondary structure by circular dichroism spectroscopy.The results showed that the secondary structure and nature of hPEPT2(560-664) would bechanged in the different pH buffer.