STUDIES WITH DNA-CELLULOSE CHROMATOGRAPHY .I. DNA-BINDING PROTEINS FROM ESCHERICHIA COLI
STUDIES WITH DNA-CELLULOSE CHROMATOGRAPHY .I. DNA-BINDING PROTEINS FROM ESCHERICHIA COLI
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DOI：
10.1101/sqb.1968.033.01.033
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发表时间：
1968-01-01
期刊：
影响因子：
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通讯作者：
FERRIS, FL
中科院分区：
文献类型：
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作者：
ALBERTS, BM;AMODIO, FJ;FERRIS, FL
为了使诸如DNA复制和修复、DNA重组、选择性基因表达和mRNA转录等基本遗传过程成为可能，细胞内的许多蛋白质必须与DNA相互作用。在基因功能可以在分子水平上精确定义之前，许多这样的DNA相关蛋白质必须单独分离和表征。我们已经制定了一个通用的方法，应有助于这种分析。这种方法，我们称之为“DNA-纤维素色谱法”，依赖于这样一个事实，即细胞内许多对DNA起作用的蛋白质在体外以生理离子强度与DNA紧密结合。在较高的盐浓度下，这些蛋白质从DNA中可逆地释放出来，显然处于未受损状态。具有这种结合特性的先前表征的蛋白质包括E.大肠杆菌RNA聚合酶（JR Richardson，1966 b; Pettijohn和Kamiya，1967）以及乳糖和噬菌体J1阻遏物（吉尔伯特和Muller-Hill，1967; Ptashne，1967 b）。这在NaCl浓度不低于0.05 M时进行。随后用相同离子强度的缓冲液洗涤柱，只留下对DNA-纤维素具有高亲和力的结合蛋白质。然后增加NaCl浓度以从DNA中分离这些蛋白质。
A fascinating variety of proteins must interact with DNA within the cell in order to make possible such basic genetic processes as DNA replication and repair, DNA recombination, selective gene expression, and mRNA transcription. Before gene function can be precisely defined at the molecular level, many such DNA-associated proteins will have to be individually isolated and characterized. We have developed a general method which should facilitate such analyses. This method, which we call'DNA-cellulose chromatography', relies upon the fact that many of the proteins which function on DNA inside the cell bind tightly to DNA at physiological ionic strengths in vitro. At higher salt concentrations these proteins are reversibly released from the DNA, apparently in an undamaged state. Previously characterized proteins with such binding properties include the E. coli RNA polymerase (JR Richardson, 1966b; Pettijohn and Kamiya, 1967) and the lactose and phage Jl repressors (Gilbert and Muller-Hill, 1967; Ptashne, 1967b).In order to isolate all DNA-binding proteins from an organism, DNA-free crude extracts are prepared and passed through a column consisting of DNA adsorbed onto an inert cellulose matrix ('DNA-cellulose'). This is done at NaC1 concentrations not lower than 0.05 M. Subsequent washing of the column with buffer of the same ionic strength leaves bound only proteins with a high affinity for the DNA-cellulose. The NaC1 concentration is then increased to elate these proteins from the DNA.