title: [The cloning, expression and the binding ability with TRβ1 of retinoid X receptor-α gene].
abstract: Retinoid X receptor-α (RXR-α), a member of nuclear receptor family, is capable of mediating retinoid signaling pathways and plays a critical role in regulating target gene transcription. To further study the function of RXR-α, abundant of recombinant RXR-α protein in hand is necessary. In this study an intact RXR-α coding sequence was amplified by RT-PCR and subsequently inserted into expression plasmid vector pQE-30Xa to form the recombinant construct of pQE-30Xa/RXR-α. Thereafter, competent bacteria Escherichia coli M15 [PREP4] was transformed and the expression of RXR-α was induced by adding IPTG to the medium. Bacterially expressed recombinant RXR-α was purified by Ni-NTA affinity chromatography and verified by SDS-PAGE and Western blotting analyses. The results showed that a protein, with the molecular mass around 50 kDa, could be selectively recognized by anti-RXR-α antibody. Co-immunoprecipitation assay indicated that this recombinant RXR-α could effectively bind TRβ1 to form a heterodimer, which could specifically bind the target DNA fragment. This was confirmed by EMSA. In conclusion, the recombinant human retinoid X receptor-α was prepared successfully, which makes a basic for further study of its function.
authors.affiliation: Key Laboratory of Hormone and Development of Tinajin City, Institute of Endocrinology/Metabolic Disease Hospital, Tianjin Medical University, Tianjin 300070, China. zhangying5883@yahoo.cn
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.forename: Li-Ling
authors.initials: LL
authors.lastname: Hu
authors.forename: Wei
authors.initials: W
authors.lastname: Xie
authors.forename: Pei
authors.initials: P
authors.lastname: Sun
authors.forename: Ai-Jun
authors.initials: AJ
authors.lastname: Zuo
authors.forename: Jing-Yu
authors.initials: JY
authors.lastname: Zhang
mesh_terms: D002460:Cell Line; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006801:Humans; D047468:Immunoprecipitation; D009571:Nitrilotriacetic Acid / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D015183:Restriction Mapping; D047490:Retinoid X Receptor alpha / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D037042:Thyroid Hormone Receptors beta / Q000737:chemistry / Q000378:metabolism*
chemical_list: D047490:Retinoid X Receptor alpha; D037042:Thyroid Hormone Receptors beta; D009571:Nitrilotriacetic Acid