Taylor I, Patel J, Firman K, Kneale G
Biophysics Laboratories, School of Biological Sciences, Portsmouth Polytechnic, UK.
Nucleic Acids Res. 1992 Jan 25;20(2):179-86. doi: 10.1093/nar/20.2.179.
Large scale purification of the type I modification methylase EcoR124 has been achieved from an over-expressing strain by a two step procedure using ion-exchange and heparin chromatography. Pure methylase is obtained at a yield of 30 mg per gm of cell paste. Measurements of the molecular weight and subunit stoichiometry show that the enzyme is a trimeric complex of 162 kDa consisting of two subunits of HsdM (58 kDa) and one subunit of HsdS (46 kDa). The purified enzyme can methylate a DNA fragment bearing its cognate recognition sequence. Binding of the methylase to synthetic DNA fragments containing either the EcoR124 recognition sequence GAAN6RTCG, or the recognition sequence GAAN7RTCG of the related enzyme EcoR124/3, was followed by fluorescence competition assays and by gel retardation analysis. The results show that the methylase binds to its correct sequence with an affinity of the order 10(8) M-1 forming a 1:1 complex with the DNA. The affinity for the incorrect sequence, differing by an additional base pair in the non-specific spacer, is almost two orders of magnitude lower.
通过两步法，利用离子交换和肝素色谱，从过表达菌株中实现了I型修饰甲基化酶EcoR124的大规模纯化。每克细胞糊可获得30毫克纯甲基化酶。分子量和亚基化学计量的测量表明，该酶是一种162 kDa的三聚体复合物，由两个HsdM亚基（58 kDa）和一个HsdS亚基（46 kDa）组成。纯化后的酶能够甲基化带有其同源识别序列的DNA片段。通过荧光竞争分析和凝胶阻滞分析，研究了甲基化酶与含有EcoR124识别序列GAAN6RTCG或相关酶EcoR124/3的识别序列GAAN7RTCG的合成DNA片段的结合情况。结果表明，甲基化酶以10(8) M-1的亲和力与其正确序列结合，并与DNA形成1:1复合物。对在非特异性间隔区相差一个额外碱基对的错误序列的亲和力几乎低两个数量级。
