title: Expression of human peroxisome proliferator-activated receptors ligand binding domain-maltose binding protein fusion protein in Escherichia coli: a convenient and reliable method for preparing receptor for screening ligands.
abstract: Human peroxisome proliferator-activated receptors (hPPARs) are ligand-activated transcription factors and are the target for the treatment of many diseases. Screening of their ligands is mainly based on assays of ligand binding to the ligand binding domain (LBD) of hPPARs.However, such assays are difficult because of the preparation of hPPARs LBD. In order to yield functional hPPARs LBD for screening ligands, hPPARs LBD was fused with maltose-binding protein(MBP) using the pMAL-p2x expression system through the gene engineering technique. The radioligand binding assay showed that MBP did not affect ligand binding with hPPARs LBD in the fusion proteins, which means that MBP-hPPARs LBD can be used instead of hPPARs LBD in ligand screening work. The results show that the new strategy using MBP as a fusion tag for preparing hPPARs LBD for screening ligands is a convenient and reliable method. It may be used to easily obtain the other nuclear receptors.
authors.affiliation: Division of Pharmacology, Chongqing Medical University, Chongqing, People's Republic of China.
authors.forename: Changqing
authors.initials: C
authors.lastname: Li
authors.forename: Mi
authors.initials: M
authors.lastname: Tian
authors.forename: Ye
authors.initials: Y
authors.lastname: Yuan
authors.forename: Qinxin
authors.initials: Q
authors.lastname: Zhou
mesh_terms: D000688:Amylose / Q000737:chemistry; D016718:Arachidonic Acid / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000473:pathology; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D018076:DNA, Complementary / Q000138:chemical synthesis / Q000235:genetics; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000378:metabolism*; D015951:Factor Xa / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008024:Ligands; D008297:Male; D058619:Maltose-Binding Proteins; D047492:Peroxisome Proliferator-Activated Receptors / Q000187:drug effects / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors
chemical_list: D002352:Carrier Proteins; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D008024:Ligands; D058619:Maltose-Binding Proteins; D047492:Peroxisome Proliferator-Activated Receptors; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D016718:Arachidonic Acid; D007544:Isopropyl Thiogalactoside; D000688:Amylose; D015951:Factor Xa