Preparation Of Human PPARγ₁LBD Recombination Protein And Screening Nature Ligands

Objective: To obtain hPPARγ1 protein with high purity and bioactivi- ty by gene recombination technique and molecular biochemistry approa- ches for screening nature hPPARγ1 ligands from traditional chinese herbs.Methods:1. A cDNA-encoding ligand binding domain (LBD) of hPPARγ1 was prepared and amplified using mRNA extracted from human fatty tissue by RT-PCR and the product was digested and inserted into the downstream of the malE gene of the vector pMAL-p2X, which encoded maltose-binding protein (MBP).2.The recombinant plasmid containing MBP-hPPARγ1 gene was transformed into E.coli. TB1 and expressed MBP-hPPARγ1LBD fusion protein under the optimized induction conditions. The host bacteria were lysed by ultrasonic treatment and centrifuged. The fusion protein in the supernatant was purified through amylose-resin affinity chromatography and digested by Factor Xa, then separated by amylose-resin affinity chromatography and DEAE-52 anion exchang chromatography. 3.Functions of MBP-hPPARγ1LBD and hPPARγ1LBD were charac- terized by radioligand saturation binding assay.4.Sreening nature ligands for hPPARγ1 was taken by radioligand com- petition binding assay.Results:1. The DNA strap with 909bp was presented after recombinant plasmid was digested by HindⅢand BamHⅠ.2.The high efficient expression of MBP-hPPARγ1 fusion protein(MW80KD, 20 mg/L culture medium) in TB1 cells was observed when 0.4 mmol/L IPTG and 6 h incubation were taken at 30℃. High pure hPPARγ1LBD (5 mg/L culture medium) was prepared after MBP-hPPARγ1 was purified by amylose-resin affinity chromatography and Factor Xa.3.The radioligand saturation binding assay showed that both MBP-hPPARγ1 and hPPARγ1 could well bind with H3-ROS (Kd=89 nmol/L), which had demonstrated both of them had biological functions and MBP didn't affect ligand to bind with hPPARγ1 in the fusion protein.4.CUR was identified to be a nature ligand for hPPARγ1 (IC50=8.82±0.74μmol/L,Ki=0.680μmol/L) among BER,CAP,CUR,DAI,EGCG,NAG,NAR,POL and RB1.Conclusions:A large amount of soluble hPPARγ1LBD protein with high purity and bioactivity has been obtained, which has been applied for screening nature ligands successfully.