猪末端转脱氧核苷酸酶的分离纯化

末端转脱氧核苷酸酶催化脱氧垓苷三磷酸连接到单链多聚脱氧核苷酸的3′—OH末端。猪胸腺匀浆抽提液,经磷酸纤维素吸附层析,硫酸铵分级沉淀,DEAE—纤维素负吸附,葡聚糖凝胶G100过滤和两次羟基磷灰石柱层析,分离后得到该酶,纯化倍数达2736倍。葡聚糖凝胶G100过滤分离出三个活力峰,用SDS-聚丙烯酰胺凝胶电泳显示其分子量分别为60000,53000和32000道尔顿,其中53000组分比活最高,达每毫克蛋白3135活力单位。

ISOLATION ANE PURIFICATION OF TERMINAI DEOXYNUCLEOTIDYL TRANSFERASE FROM HOG THYMUS

Terminal deoxynucleotidyl transferase (TdT) that catalyzes the addition of deoxynueleoside triphosphate on the 3'-OH end of the single-stranded polydeoxynucleotide primer was purified 2736-fold from Crude extract of hog thymus, Purification procedures included ammonium sulfate precipitate, chromatographic separations on phosphocellulose and DEAE-cellulose colums, Sephadex G-100 gel filtration and hydroxyapatide chromatography. Three active fractions were isolated, and the molecular weights were estimated to be about 60000, 53000 and 32000 Daltons respectively by SDS-polyacrylamide gel electrophoresis. The specific activity of 53000 fraction was found to be 3135u/rag protein.