Study On The Functional Petides Of Oligopeptide Transporter Protein PepT2

PepT2is a member of the POT transporter family, mainly exists in the kidneys, lungs,brain, and the epithelial cells of breast’s bottom. The substrates of PepT2are dipeptide,tripeptide and some peptide drugs. PepT2is composed of729amino acids containing12transmembrane domains which contain a number of conserved amino acids sequences. Theseconserved amino acid sequences may be necessary to transporting of protein, so the study onthe characteristics of these conservative sequence will provide the foundation for researchesabout the oligopeptide transport protein.Through the segmentation method, PET30a(+)/PepT2(281-362) were obtained by usingPCR amplification technology, and transformed into E. coli BL21(DE3) in order to get theexpression system of PET30a(+)/PepT2(281-362)/BL21(DE3). Then IPTG induction expressconditions were explored in the expression system PET30a(+)/PepT2(1-174)/BL21(DE3) bySDS-PAGE gel electrophoresis method, and the results showed that the induction express ofPepT2(1-174) was the most stable at37°C with3h and0.5mmol/L IPTG.Conservative peptide FYLSINAGS located in PepT2(286-294) and four its derivativesFYGLINSGG, FYGLINKGG, FYGLINAGG, FYLSINAGG were prepared by solid phasesynthesis, the products were separated and purified using HPLC, and identified by using massspectrometry. At last, effects of factors, such as reaction time and peptides concentration, onthe interaction between peptides and dipeptides YY, as well as between peptides and DNAwere researched by using UV and fluorescence spectrometry. The results showed that theeffect of reaction time on the interaction was minor. Except the FYLSINAGG, the UVabsorption of all the other systems hyperchromic then underwent hypochromic effect. Thefluorescence quenching process of dipepides YY and DNA-EB system was proven a staticone after the determination of its quenching constant. Comparisons between the UV andfluorescence spectrum suggested that serine is a more important functional amnio acid thanglycine in conserved sequence, ensuring a higher activity of peptide when located in theC-end.