Kelly S, Kaddurah-Daouk R, Smith H O
J Biol Chem. 1985 Dec 5;260(28):15339-44.
An Escherichia coli K12 strain carrying the HhaII methylase and restriction genes on two separate compatible plasmids, pSK5 and pSK7, is used to overproduce the restriction endonuclease. Plasmid pSK5 expresses the methylase gene constitutively from its chloramphenicol resistance gene promoter, and plasmid pSK7 expresses the restriction endonuclease under control of the lacUV5 promoter. Induction of the two-plasmid clone with 1 mM isopropyl-1-thio-beta-D-galactopyranoside results in a 15-fold increase in HhaII endonuclease activity. The enzyme has been purified to apparent homogeneity. It migrates as a 23-kilodalton polypeptide on denaturing sodium dodecyl sulfate-polyacrylamide electrophoretic gels and as a 52-kilo-dalton native protein dimer on a high pressure liquid chromatography sizing column.
一株携带HhaII甲基化酶和限制基因的大肠杆菌K12菌株，这两个基因分别位于两个相容的质粒pSK5和pSK7上，用于过量生产限制性内切酶。质粒pSK5从其氯霉素抗性基因启动子组成型表达甲基化酶基因，质粒pSK7在lacUV5启动子的控制下表达限制性内切酶。用1 mM异丙基-1-硫代-β-D-吡喃半乳糖苷诱导双质粒克隆，可使HhaII内切酶活性增加15倍。该酶已纯化至表观均一。在变性十二烷基硫酸钠-聚丙烯酰胺电泳凝胶上，它作为一条23千道尔顿的多肽迁移，在高压液相色谱尺寸排阻柱上作为一个52千道尔顿的天然蛋白质二聚体迁移。
