Expression Of Peptide Transporter PEPT2 And Modification Of Ferulic Acid

PepT(PepT1:SLC15A1 and PepT2:SLC15A2)can transport di-peptides and tri-peptides widly. The mechanism of PepT2-mediated transportation depend on transport system of H+ and Na+/H+ pump with non-enegry consumption to work inversing to concentration gradients. PepT2 is a high-affinity and low-capacity transporter that is widely distributed in the body to transport di-peptides and tri-peptides, as well as various peptidyl drugs such asβ–lactam antibiotics angiotensin-converting enzyme inhibitors and bestatin. It is demonstrat that mechanism of PepT2-mediated transportation is different from the absorption of amino acids.The sequence of PepT2 DNA was analysied using the software of Blast(NCBI), Bioediter in this paper. Then a pair of primers was designed for the amplification of peptide transporter gene of human using PCR method. A recombinant plasmid pET30a(+)/PepT2 was constructed, and then this recombinant plasmid was converted into Escherichia coli BL(DE3)pLYS. The results of sequencing showed that three bases mutations of PepT2 gene were found after PCR(A672T,A966T,G1916A), and one base mutation lead to amino acid mutation(G640S). The plasmid was induced to express the recombinant protein in Escherichia coli. The results showed that the protein could not be detected that may be caused by the rare bases which was up to 11% in the full gene length. The gene should be optimized for the further expression.Two ferulic acid amide derivatives were synthesized in liquid phase way: acetylferulic– phenylalanine methyl ester, acetylferulic–tyrosine methyl ester. Two derivatives were synthesized in solid-phase way: acetylferulic–tyrosine; acetylferulic–tyr–tyrosine(F-Tyr-Tyr). The synthesized ferulic acid amide derivatives were characterized by IR, ESI-MS, 1HNMR and 13CNMR. The interaction between ferulic acid/ F-Tyr-Tyr and DNA were investigated by UV and fluorescence spectroscopy. The effects of different factors on the interaction were studied, including different concentrations of ferulic acid/ F-Tyr-Tyr, action time and tempreture. The absorption spectra of Ferulic-DNA system showed hypochromicity, indicating that the interaction between Ferulic and DNA might be in electrostatic binding mode. The fluorescence of EB-DNA had quenching effect with increasing the concentration of Ferulic acid, and the quenching constant was 1.015×103L·(mol·s)-1. The Stern-Volmer equation was indicated that the quenching process was electrostatic bonding . The absorption spectra of F-Tyr-Tyr-DNA system showed hypochromicity, indicating that the interaction between F-Tyr-Tyr and DNA might be in electrostatic binding mode. The fluorescence of EB-DNA had quenching effect with increasing the concentration of F-Tyr-Tyr-DNA, and the quenching constant was 6.28×10³L·(mol·s)^-1. The Stern-Volmer equation was indicated that the quenching process was electrostatic bonding .