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Peroxisome proliferator-activated receptors(PPARs) are ligand-activated transcription factors known to play a pivotal role in regulations of metabolism.In order to yield soluble ligand binding domain of PPARδ(PPARδLBD) for screening ligands,the cDNA was amplified using total RNA from HepG2 cells by RT-PCR.Then the enzyme-digested product was inserted downstream of the malE gene in the vector pMAL-p2x,which encoded maltose-binding protein(MBP),resulting in the expression of an MBP-PPARδLBD fusion protein.The recombinant plasmid was transformed into E.coli TB1 that was cultured shakily at 30 °C,200 r/min and induced by 0.4 mmol/L IPTG for 6 h.The cells were harvested by centrifugation and broken by sonication.The expressed fusion protein was soluble and accounted for 0.31 of the total protein in the supernatant.Western blot analysis showed that the expressed MBP-PPARδLBD could bind to anti-MBP-antibody.The MBP-PPARδLBD fusion protein of 77 kDa and the PPARδLBD protein of 34 kDa were obtained by amylose-resin affinity chromatography without or with digestion of Factor Xa.They were both homogeneity,judged by SDS-PAGE.The recombinant MBP-PPARδLBD and PPARδLBD protein with high purity is obtained,which provides the necessary material for screening and researching PPARδ ligands.
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors known to play a pivotal role in regulations of metabolism. In order to yield soluble ligand binding domain of PPARδ (PPARδ LBD) for screening ligands, the cDNA was amplified using total RNA from HepG2 cells by RT-PCR. The enzyme-digested product was inserted downstream of the malE gene in the vector pMAL-p2x, which encoded maltose-binding protein (MBP), resulting in the expression of an MBP-PPARδ LBD fusion protein. The recombinant plasmid was transformed into E. coli TB1 that was cultured shakily at 30 ° C, 200 r / min and induced by 0.4 mmol / L IPTG for 6 h. The cells were harvested by centrifugation and broken by sonication. expressed fusion protein was soluble and accounted for 0.31 of the total protein in the supernatant. Western blot analysis showed that the expressed MBP-PPARδ LBD could bind to anti-MBP-antibody. The MBP-PPARδ LBD fusion protein of 77 kDa and the PPARδ LBD protein of 34 kDa we re obtained by amylose-resin affinity chromatography without or with digestion of Factor Xa.They were both homogeneity, judged by SDS-PAGE. recombinant MBP-PPARδLBD and PPARδLBD protein with high purity is obtained, which provides the necessary material for screening and researching PPARδ ligands.