The Preliminary Studies On A Novel Thyroid Hormone Receptor β Isoforms

Thyroid hormone receptors(TRs)belong to the nuclear hormone receptor superfamily.TRs are coded by two genes,TRαand TRβ.They separately coded two receptor subtypes.Up to date ten isoforms has been identified because of different transcription start position and different splicing.TRαcoded TRα,TRα2,TRα3,TRΔα1,TRΔα2.TRβcoded TRβ1,TRβ2,TRβ3,TRΔβ3 and a novel isforms TRβΔ^* which was found by our laboratory.We have found this novel isoforms of TR in the Rat liver.It is the unique extended- pattern TR isoforms,and extension of its sequence is between exon 3 and 4 of TRβ.It is possible that there is a new exon within intron 3-4.In this study,our purpose was to express this new isforms of thyroid hormone receptor protein in E.coli and achieve the high expression of this receptor in E.coli. Then we aimed to prove the correctness of amino acid sequence deduced from the mRNA by amino acid composition analysis of purified protein.The dynamic analysis of binding T3 and the EMSA of binding target DNA as some biological activities of the receptor was also determined.Firstly,we got the total sequence of TRβΔcDNA from pGEM-T Easy/TRβbuilt recombinant plasmid.The target sequence was inserted into prokaryotic fusion expression vector pETDuet-1,tested by DNA sequencing.Then recombinant plasmid pETDuet-1/TRβΔwas transformed into E.coli BL21(DE3)and expressed.The amount and molecular weight of the recombinant protein were tested by SDS-PAGE.Western blotting revealed that the 58-KD protein was recombinant TRβΔ.After the culturing conditions were optimized,the yield of TRβΔwas 21.94mg/L and accounted for 32.00%of total protein in E.coli BL21(DE3).Electrophoretically pure protein was obtained by a fast electrophoretic method for elution of fusion protein in dialysis bag.Amino acid analyzer was used for quantitative analysis of amino acid composition of the protein.The target proteins containing 6×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.Finally,by radioligand binding assay(RLBA),recombinant TRβΔwas identified to have biological activity of binding T₃.