Establishment Of HPPARα Ligand Screening Platform By HPLC-UV Method

Aim To establish a screening platform for ligands of human peroxisome proliferator-activated receptor alphaMethods Under the optimized induction conditions, the host bacteria harbouring the recombinant plasmids(pMAL-p2X-hPPARs/TB1) were lysed by ultrasonic treatment and then centrifuged. The MBP-PPARsLBP fusion proteins in the supernatant were prepared through amylose-resin affinity chromatography, or digested by the protease factor Xa and purified with DEAE-52 anion exchange chromatography for preparation PPARsLBD with high purity. The HPLC conditions were as follows: chromatographic column of HiTrap desalting (SephadexG-25,1.6×2.5cm), mobile phase of PBS, column temperature of 8℃, sample size of 20μL and elution at flow rate of 1 ml/min. First of all,technology investigation was carried out through detecting bezafibrate (Bez). Then, the reaction conditions of binding were optimized. Binding assay of Bez with different concentrations to receptor protein was carried out using rosiglitazone (Ros) as a negative drug. Under optimized conditions the specific binding (SB) of Bez was calculated. The judgement standard of screening hPPARs ligand by HPLC-UV was established on the basis of experimental results. Finally, the validity of ligand screening platform was tested by drugs that either were selective agonists of hPPARαsuch as Fen and Bez , or were selective agonists of hPPARr such as Ros and Pio.Results The host bacteria harbouring the recombinant plasmids were induced under the optimized conditions and one liter culture could yield approximately 20 mg . The yields of MBP-PPARsLBD and PPARsLBD were about 14 mg and 5 mg per liter of culture, respectively, through the purification procedure. The retention time of PPARs was 1.6 min , Bez was 6.3 min at the chromatogram with good seletivity, high column efficiency, and symmetry peak shape. A good linear range of Bez fell in 0.5-200μmol / L, LOD was 0.2μmol / L, RSD was less than 5% in days or day. The SB / TB values of Bez were more than 65% under the best optimization of reaction conditions no matter MBP-hPPARαLBD or hPPARαLBD was used. According to Molecular Exclusion Chromatography (MEC) theory and binding assay results with Bez and Ros, we considered that the hPPARαligand screening platform was established using HPLC-UV method. The KD of Bez was 0.91μmol/L .Conclusions The HPLC-UV method is a convenient and simple method for screening hPPARαligand.