Prokaryotic Expression Of PepT2and Modification Of L-dopa

Human peptide transporter （hPepT2, SLC15A2） is one of the peptide transporters, whichis responsible for the reabsorption of di-and tri-peptides in kidney. PepT2protein consists of729amino acids, and contains12transmembrane domains. In addition to small peptides,some peptide-like drugs, including angiotensin-converting enzyme inhibitors, anti-viral andanti-cancer drugs are also the substrate of PepT2. To improve the bioavailability ofmedicaments, more and more attention has been given to the designing of PepT2targetingpharmaceuticals. Gene expression technology is a popular means to express functionalproteins. E. coli expression system is an important tool commonly used in gene expressiontechnology. But there are some rare codons in eukaryotic gene that can’t be recognized andexpressed by prokaryotic expression system. So we tried to express the segment of hPepT2without rare codons in prokaryotic expression system to investigate the function of thesefragments.hPepT2（1-174） gene and hPepT2（624-729） gene were amplified by PCR, and thenrecombined into plasmid pET30a（+） and pUC18. The recombinant plasmid pET30a（+）/hPepT2（1-174） and pUC18/hPepT2（624-729） were transformed into E. coli BL21（DE3）.Prokaryotic expression systems pET30a（+）/hPepT2（1-174）/BL21（DE3） and pUC18/hPepT2（624-729）/BL21（DE3） were constructed successfully. Then SDS-PAGE was applied tocontain the ITPG induced conditions. The yield of segment protein is stable after induced for4hours.Three acid amide derivatives of L-dopa were synthesized by Fmoc solid-phase synthesis.The crude products were purified through high performance liquid chromatographytechnology, and the derivatives were characterized by ESI-MS,¹HNMR and¹³CNMR. Theinteractions between acid amide derivatives of L-dopa and Calf thymus DNA were studied byUV and fluorescence spectroscopy. The interactions between acid amide derivatives ofL-dopa with pUC18plasmid DNA were identificated by agarose gel electrophoresis. Withincreasing the concentration of acid amide derivatives of L-dopa, the UV absorption spectraof system showed hypochromicity, showing that the interaction mode between acid amidederivatives of L-dopa and ctDNA might be hydrophobic interaction and intercalationinteraction. With increasing the concentration of acid amide derivatives of L-dopa in system, the fluorescence of the system all showed quenching effect. According the Stern-Volmerequation, the quenching constants were calculated. All the quenching constants were higherthan the collision constant of the largest diffusion between small molecules and biologicalmacromolecules, indicating that the quenching process was a static mode. With increasing theconcentration of acid amide derivatives of L-dopa in system, the moving speed of pUC18became slower, showing the intercalation interaction exist between derivatives of L-dopa andpUC18DNA. The experimental results showed that the interaction between these threepeptide derivatives of L-dopa and DNAwere stronger compared with L-dopa, and with thegrowth of peptide chain, the interaction between the compound and DNA increases.