摘要
通过硫酸铵盐析,DEAE-纤维素柱层析,磷酸纤维素亲和层析及SephadexG-100凝胶过滤法,从噬淀粉芽孢杆菌HI(Bacillus amyloliguefaciens HI)提纯了DNA甲基化酶。用聚丙烯酰胺凝胶电泳检查,已达电泳均一,比活力提高了326倍。并用聚丙烯酰胺梯度凝胶电泳和Sephadex G-100凝胶过滤法测得其天然酶的分子量为273000,又用SDS聚丙烯酰胺凝胶电泳测得它的亚基分子量为34500,故该酶有8个分子量相同的亚基。用凝胶电聚焦法测得其pI_(22 c)=9.0。
A simple and efficient procedure for purifying Bam HI DNA methylase has been established. The purification steps include. 1, Disruptoing of bactreial cell by ultrasonication; 2, Remove the endogenous DNA by streptomycin sulfate; 3, Salting out by(NH4)2SO4; 4, Ion exchange chromatography on DEAE-cellulose (DE-52); 5, Affinity chromatography on phosphocellulose (P11)} 6, Gel filtration over Sephadex G-100.The DNA methylase from Bam HI (Bacillus amyloliquefaciens) has been purified for 326 folds by this procedure. The electrophotogram of the DNA methylase preparation revealed a single band on PAGE and pore gradient PAGE. The enzyme has a molecular weight of 272 000 as determined by gel filtration on Sephadex G-100 and PG-PAGE. The result of SDS-PAGE reveals that this enzyme is made of a sigle type of subunits,and the molecular weight of the snbunit is 34900. Therefore this enzyme consists of eight identical subunits The pI(22℃) of this enzyme is 9.0 as showon by IEF-PAGE.
基金
国家自然科学基金(高技术)资助项目。项目号:0388011~~
关键词
DNA甲基化酶
提纯
理化性质
Chromatography, Electrophoresis, Molecular Weight, Isoelectric Point (pI), Subunit
