Prokaryotic Expression And Properties Of Human Oligopeptide Transporter PepT2(579-664)

Nutrition researches show that protein is absorbed mainly in the form of small peptidesthrough transported by oligopeptide transporters in human body. Proton-coupled oligopeptidetransporters family is comprised by PepT1, PepT2, PHT1and PHT2, responsible fortransport of most dipeptide, tripeptide and peptide-like drugs in human body. In studiedliteratures, PepT2is widely expressed in a variety of tissues, with predominant expression inthe kidney but also in brain, eye, mammary gland, and so on, responsible for the reabsorptionof peptide and peptidomimetics. hPepT2contains12transmembrane domains （TMD） and anextracellular loop between9TMD and10TMD, has been characterized as a high-affinity,low-capacity transporter. The analysis of chimeric PepT1/PepT2proteins indicate that thephenotypic characteristics of PepT2were determined by1-9TMDs, the putative substratebinding domains in PepT2lie in the region7,8, and9TMDs, and the functions of last threeTMDs were unknown.First of all, prokaryotic expression of hPepT2（579-664） was carried out in EscherichiaColi BL21（DE3）pLysS. Here we have cloned the gene of hPepT2（579-664） by using PCR,and linked it with pET30a（+）. Then pET30a（+）-hPepT2（579-664） recombinant plasmid wastransformed into BL21（DE3）pLysS for expression, and induced by0.3mM IPTG at37℃for4h, and the expression product was detected by15%SDS-PAGE. The result showedhPepT2（579-664） had been expressed a certain amount. Cells were harvested and thenwashed with PBS lysis buffer. Then crude protein was obtained after disrupted the bacteriawith sonication, and target protein was abtained through purfied crude protein by gelchromatography with SephadexTM G-75medium. The solubility of hPepT2（579-664） wasstudied with UV-Vis spectrophotometer; the influence of different solution pH on stability ofhPepT2（579-664） was studied by using UV-Vis spectrophotometer and fluorescencespectroscopy. The results indicated that hPepT2（579-664） was stable at pH7.5.Ten dipeptides were synthesized in solid-phase way: Tyr-Gly, Tyr-Leu, Tyr-Phe, Tyr-lys,Tyr-Arg, Tyr-Tyr, Ser-Tyr, Pro-Tyr, Val-Tyr, and β-Ala-Tyr. The synthesized products wereanalyzed and purified by RP-HPLC and characterized by ESI-MS. The interaction betweendipeptides and protien were investigated by fluorescence spectroscopy. The effects ofdifferent concentrations and reaction time of dipeptides on the interaction were studied. Theresults obtained showed that protein structure can be changed by dipeptides, the concentration of protein was2.52mg/ml and its fluorescence intensity was strongest when the concentrationof dipeptide solution was3.33×10^-3-1.0×10^-2mg/ml. The fluorescence intensity of proteinwas gradually increased as the increase of reaction time, and equilibrium after10h. The datasuggested that hPepT2（579-664） can be interacted with dipeptides, which will providedgroundwork for function reseachs and substrate structures reseaches of human PepT2and thedesign of the new drugs.