A novel strategy for the expression and purification of the DNA methyltransferase, M-AhdI
A novel strategy for the expression and purification of the DNA methyltransferase, M-AhdI
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DOI：
10.1016/j.pep.2004.06.008
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发表时间：
2004-09-01
影响因子：
1.6
通讯作者：
Kneale, G
中科院分区：
文献类型：
--
作者：
Marks, P;McGeehan, J;Kneale, G
从类型1 1/2 R-M系统的甲基酶的生化和结构研究AhdI要求有能力以可溶性和活性形式大量纯化这种多亚单位酶。对几种大肠杆菌表达系统生产完整甲基酶的能力进行了测试，但这不能在简单的共表达系统中实现。对表达实验进行了优化，获得了高产量的可溶性M亚基和S亚基作为单独的蛋白质。温度和诱导条件被证明是最有用的因素，尽管S亚基的纯化成功，但对M亚基的有效策略仍然缺乏。一种新的策略被开发出来，在这种策略中，单个亚基被单独表达，细菌细胞在裂解前混合。该方法通过肝素和大小排斥层析将多亚单位甲基酶纯化为均一组分，产量较高。然而，在1M的氯化钠中，通过硫酸铵沉淀去除紧密结合的DNA是必要的。现在可以始终如一地生产完整的甲基酶，避免使用融合蛋白。与单个亚基不同，纯化的酶在很长一段时间内都是稳定的。这种方法可能在多亚基蛋白的表达或其个别成分的表达有问题的情况下普遍应用。(C)2004 Elsevier Inc.保留所有权利。
Biochemical and structural studies of the methylase from the type 1 1/2 R-M system AhdI require the ability to purify this multisubunit enzyme in significant quantities in a soluble and active form. Several Escherichia coli expression systems were tested for their ability to produce the intact methylase but this could not be achieved in a simple co-expression system. Expression experiments were optimised to produce high yields of soluble M and S subunits as individual proteins. Temperature and conditions of induction proved to be the most useful factors and although purification of the S subunit was successful, an efficient strategy for the M subunit remained elusive. A novel strategy was developed in which individual subunits are expressed separately and the bacterial cells mixed before lysis. This method produced a high yield of the multi-subunit methylase when purified to homogeneity by means of heparin and size-exclusion chromatography. It was found to be essential, however, to remove tightly bound DNA by ammonium sulphate precipitation in 1 M NaCl. The intact methylase can now be consistently produced, avoiding the use of fusion proteins. The purified enzyme is stable over long time periods, unlike the individual subunits. This method may be of general application where the expression of multi-subunit proteins, or indeed their individual components, is problematic. (C) 2004 Elsevier Inc. All rights reserved.