Purification of overexpressed gam gene protein from bacteriophage Mu by denaturation-renaturation techniques and a study of its DNA-binding properties.
Purification of overexpressed gam gene protein from bacteriophage Mu by denaturation-renaturation techniques and a study of its DNA-binding properties.
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DOI：
10.1042/bj2690679
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发表时间：
1990-08
期刊：
影响因子：
--
通讯作者：
Z. Abraham;N. Symonds
中科院分区：
文献类型：
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作者：
Z. Abraham;N. Symonds
在大肠杆菌中合成的重组Mu Gam基因蛋白（Mu GAM）以不溶性包涵体的形式积累，在细胞裂解和低速离心后，可以在沉淀级分中回收。这种性质被用于Mu GAM的纯化程序中，该程序基于盐酸胍变性-复性，然后进行单一的DEAE-纤维素色谱步骤。纯化的穆GAM硝化纤维素过滤器结合实验表明，以高亲和力结合线性双链DNA和更弱的超螺旋和单链形式。Mu GAM保护线性DNA免于被多种核酸外切酶降解，但仅微弱地抑制核酸内切酶活性。这些结果与Mu GAM通过结合DNA末端而赋予外切核酸酶活性保护的模型雅阁。
Recombinant Mu gam gene protein (Mu GAM) synthesized in Escherichia coli accumulates in the form of insoluble inclusion bodies which, after cell lysis and low-speed centrifugation, can be recovered in the pellet fraction. This property was utilized in a purification procedure for Mu GAM based on guanidine hydrochloride denaturation-renaturation followed by a single DEAE-cellulose chromatographic step. The purified Mu GAM was shown by nitrocellulose-filter-binding experiments to bind with high affinity to linear double-stranded DNA and more weakly to supercoiled and single-stranded forms. Mu GAM protects linear DNA from degradation by a variety of exonucleases, but only weakly inhibits endonuclease activity. These results are in accord with a model of Mu GAM conferring protection from exonuclease activity by binding to the ends of DNA.