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The DNA-binding of the Novel Thyroid Hormone Receptor Isoform-TRβ△

Author: HuLiLing
Tutor: ZhangJingYu;LiangDongChun;ZuoAiJun;SunZuo
School: Tianjin Medical University
Course: Biochemistry and Molecular Biology
Keywords: Thyroid hormone receptor Retinoid retinoid X receptors New subtype Co-immunoprecipitation Electrophoretic mobility shift assay
CLC: Q75
Type: Master's thesis
Year: 2010
Downloads: 21
Quote: 0
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Thyroid hormone receptor (thyroid hormone receptor, TR) is a member of the nuclear receptor superfamily, is widely distributed in various tissues, often associated with retinoid x receptor (retinoid X receptor, RXR) TRS / RXR hybrid two dimer form of specifically binding to the thyroid hormone response element within the nucleus of the target gene sequence (the thyroid hormone response elements, TRES). TR there are two main subtypes: TRα and TRβ. Tra and TRβ transcripts (transcript) due to alternative splicing or transcription initiation position different the several isoenzymes body (isoform of). In 9 subtypes the the reported rat TRS: TRα1, TRα2 TRα3, TRΔα1 TRΔα2, TRβ1, TRβ2, TRβ3 and TRAβ3, the room is also found in the study of thyroid hormone receptor in a new same reactive body, NCBI named TRβΔ mRNA compared with only TRβ1 mRNA showing a 108bp fragment having a high homology to, TRβΔ protein Also TRβ1, only near the amino terminus of more than 36 amino acids. TRβΔ protein is the only extended TRS subtypes, has a very important scientific significance. The purpose of detecting new subtype of our new discovery of a thyroid hormone receptor TRβΔ with RXR alpha, tres (DR) of the biological activity of binding, we designed the following experiment. Method 1 pETDuet-1/rRXRα recombinant expression vector. Since the room has been constructed recombinant plasmid pETDuet-1/rTRβΔ/rRXRα, Get the rRXRα the cDNA, and insert it into the prokaryotic expression vector pETDuet-1, DNA sequence analysis. (2) induced expression of the recombinant plasmid in E. coli BL21 (DE3): recombinant expression plasmid pETDuet-1/rRXRα was transformed into E. coli BL21 (DE3), 37 ° C, IPTG (1 mmol / L) induced expression of six hours, SDS -PAGE identification of the size of the expression product and the expression of Western-blot further identification. IPTG induction recombinant expression the plasmid pETDuet-1/rTRβ1 pETDuet-1/rTRβΔ, induced expression of the two products by SDS-PAGE and Western Blot identification. Immune co-precipitation (Co-immunoprecipitation, Co-IP) identification TRβ1/RXRα, TRβΔ / RXR alpha heterodimer formation. Were established TRβ1-RXRα, TRβΔ-RXRα recombinant protein complexes, each of the RXRα antibody or mouse anti-rabbit anti-His antibody was used for immunoprecipitation experiments, the precipitate to the mouse anti-His antibody or rabbit anti-RXRα antibody Western Blot analysis. 4.TRβΔ DNA binding activity of identification. TRβ1 first, TRβΔ protein were incubated at room temperature with RXRα protein, then the use of electrophoretic mobility shift test (EMSA) detection heterodimer DNA binding simultaneously detect single TRβΔ, TRβ1, RXR alpha protein tres (DR 4) a combination of circumstances. Results 1 was successfully constructed the pETDuet-1/rRXRα a recombinant expression vector. 2. Successfully expressed RXRα fusion protein: SDS-PAGE, the recombinant plasmid pETDuet-1/rRXRα transformed bacteria, pETDuet-1/TRβ1 transforming bacteria and pETDuet-1/TRβΔ of transformed bacteria-induced expression products, are in the vicinity of about 55kD deeply stained strip with (RXR alpha approximately 50kD, the TRβ1 about 52kD, TRβ △ approximately 56 kD), the size of the target protein with the expected basic, and is mainly present in the inclusion bodies. RXRα, TRβ1, TRβΔ expression were 213.8mg / L, 251.0mg / L, 161.9mg / L medium, three recombinant protein accounted for a percentage of the total bacterial proteins were 32.2%, 30.5%, 28.8%. 3 RXR alpha form of inclusion bodies, TRβ1, TRβΔ recombinant proteins soluble in high concentrations of urea solution, the supernatant after dialysis accounting for the main part. Immune co-precipitation confirmed that TRP △ and TRβ1 RXRα protein to form TRβΔ / RXR alpha heterodimer of TRβ1/RXRα. The TRβ1 TRβΔ two proteins with RXRα heterodimer formed are capable of binding to Tres (DR), but also a separate RXR alpha, TRβ1, TRβΔ proteins were also capable of binding to the TRES (DR 4). Conclusion In this paper by building pETDuet-1/rRXRα a recombinant expression vector successfully expressed RXRα protein. The results confirmed thyroid hormone receptor TRpΔ the TRβ1, and RXRα form a heterodimer with DNA, and TRβΔ, TRβ1 and RXRa three caught DNA binding activity. Our findings differ Williams reported, Williams his article, it was reported that a separate TRβ1, RXR alpha protein are not able to combine tres (DR), only the TRβ1 and RXRα were incubated form TRβ1/RXRα heterodimer before tres (DR) combined. This paper shows TRβΔ TRβ1 or RXRα alone can also be combined with the DR, but they are based on the monomer or homodimer with DNA binding needs further study.

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