Modification Of Nicotinic Acid And Expression Of Peptide Transporter PepT2(401-567)

Nicotinic acid is one of the thirteen necessary vitamins. It can be used to prevent andtreat pellagra, hypovitaminosis, angiospasm of peripheral nerve, arteriosclerosis and so on.Large dose of nicotinic acid can reduce the triglycerides of blood plasma and restrain theformation of cholesterol. Large dose of nicotinic acid can cause side effects. Peptidetransporter system of mammalian mediates the absorption of peptides and peptidyl drugswhich have amido bond. Nicotinic acid modified by amino acids as niacinamide can eliminateits side-effect. The bioavailability of drugs may be improved via peptide transporter system.In this study, nicotinic acid is modified by peptides. NA-Leu、 NA-Leu-His、NA-Asp-Asp、NA-Tyr-Tyr and NA-Asp-Asp-Asp-Asp were efficiently synthesized by solidphase method and characterized by ESI-MS,1H NMR and13C NMR.The interactions between nicotinic acid, niacinamide compounds and ct-DNA wereinvestigated by UV and fluorescence spectroscopy. The results showed that the interactionbetween NA、NA-Tyr-Tyr and ct-DNA primarily were groove mode and the binding constantswere5.81×10²L·mol^-1and7.25×10²L·mol^-1. The interaction between NA-Asp-Asp、NA-Asp-Asp-Asp-Asp、NA-Leu-His and ct-DNA primarily were groove and static modes.The binding constants were1.84×10²L·mol^-1、1.886×10²L·mol^-1and8.2×10³L·mol^-1.Theinteraction between NA-Leu and ct-DNA primarily was groove and weak insert modes. Thebinding constant was1.88×10³L·mol^-1. Those data showed that the binding constant ofniacinamide compounds which modified by acidic amino acid with ct-DNA decreased. Thebinding constant of niacinamide compounds which modified by basic and neutral amino acidswith ct-DNA increased. The binding constant decreased because of electrostatic repulsionbetween acidic amino-acid residue and phosphate of ct-DNA. The binding constant increasedbecause of electrostatic attraction between basic amino-acid residue and phosphate ofct-DNA. The influence of neutral amino-acid residue on the conjugated system of ct-DNAmade the binding constant increase.Human peptide transporter2（hPepT2） is a high-affinity and low-capacity transporter.Study on the interaction between peptidyl drugs and hPepT2has great scientific significanceand applicative value in designing and developing new peptidyl drugs. In this paper, thehPepT2（401-567） gene was amplified using PCR method. The recombinant plasmid pET30a（+）/PepT2（401-567） was constructed, and then was converted into Escherichia coliBL21（DE3）pLys. Expession of hPepT2（401-567） was induced with IPTG.. The result showedthat hPepT2（401-567） did not express in E.coli BL21（DE3）pLys. The hPepT2（401-567）should be expressed in the bacterial strain which can express rare codons or in eukaryoteexpress system.