Cloning Of Rat Thyroid Hormonr Receptor β1 CDNA And Its Expression In E.coli.

Thyroid hormone receptor (TR) is member of nuclear receptor superfamily and expressed in almost every tissue in normal body.TR has two subtypes,TR α and TR β .Because of different transcript start position and different splice,each subtype has several isoforms,including TRal,TRa2,TRa3,TRΔal,TRΔa2,TRβl, TRβ2,TRβ3,TRΔ63,etc.In clinic research,TR gene has a close relationship with resistance to thyroid hormone (RTH).And thorough study of TRβstructure is also very important for design of new blood lipid-lowering drugs.In this study,firstly,full-length sequence encoding rat TRβ1(rTRβ1) gene was obtained by RT-PCR using total RNA derived from rat liver as template, and thenit was cloned into pGEM-T Easy vector.Secondly,after corrected the wrong nucleotide,the target sequence was inserted into prokaryotic fusion expression vector pQE-30Xa,tested by DNA sequencing.Finally, recombinant plasmid pQE-30Xa/rTRβ1 was transformed into E.coli M15[pREP4] and expressed.The amount and molecular weight of the recombinant protein were tested by SDS-PAGE.Western blotting revealed that the 56-KD fusion protein was rTRβ1.After the culturing conditions were optimized,the yield of rTRBl was 6mg/L and accounted for 24.32% of total protein in E.coli M15[pREP4].The target proteins containing 6 X His affinity tag facilitates binding to Ni-NTA resin in metal chelating affinity chromatography.After the proteins binding non-specifically to resin were removed by washing solutions,the target proteins were eluted from resin with apparent higher concentration of imidazole, dialyzed and concentrated by freeze-dry.By electrophoretic mobility shift assay (EMSA) and radioligand binding assay(RLBA), recombinant rTRB1 was identified to have biological activity of binding target DNA and T₃.