AFFINITY PURIFICATION OF SEQUENCE-SPECIFIC DNA-BINDING PROTEINS
AFFINITY PURIFICATION OF SEQUENCE-SPECIFIC DNA-BINDING PROTEINS
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DOI：
10.1073/pnas.83.16.5889
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发表时间：
1986-08-01
影响因子：
11.1
通讯作者：
TJIAN, R
中科院分区：
文献类型：
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作者：
KADONAGA, JT;TJIAN, R
我们描述了一种快速有效的序列特异性DNA结合蛋白的亲和纯化方法。含有序列特异性DNA结合蛋白识别位点的互补化学合成的寡聚脱氧核苷酸被退火并连接以得到寡聚体。然后用溴化氰将该DNA共价偶联到SepharoseCL-2B上，得到亲和树脂。部分纯化的蛋白质组分与竞争对手的DNA结合，然后通过DNA-Sephose7树脂。纯化所需的序列特异性DNA结合蛋白是因为它优先与亲和树脂中的识别位点结合，而不是与溶液中的非特异性竞争对手DNA结合。例如，富含转录因子Sp1的蛋白质组分可以通过两个连续的亲和层析步骤进一步纯化500-1000倍，以30%的产率获得估计90%的同质性Sp1。此外，使用包含不同蛋白质结合位点的串联亲和色谱柱允许从同一提取物中同时纯化多个DNA结合蛋白。该方法为纯化稀有序列特异的DNA结合蛋白，如Sp1和CAAT结合转录因子提供了一种手段。
We describe a method for affinity purification of sequence-specific DNA binding proteins that is fast and effective. Complementary chemically synthesized oligodeoxynucleotides that contain a recognition site for a sequence-specific DNA binding protein are annealed and ligated to give oligomers. This DNA is then covalently coupled to Sepharose CL-2B with cyanogen bromide to yield the affinity resin. A partially purified protein fraction is combined with competitor DNA and subsequently passed through the DNA-Sepharose resin. The desired sequence-specific DNA binding protein is purified because it preferentially binds to the recognition sites in the affinity resin rather than to the nonspecific competitor DNA in solution. For example, a protein fraction that is enriched for transcription factor Sp1 can be further purified 500- to 1000-fold by two sequential affinity chromatography steps to give Sp1 of an estimated 90% homogeneity with 30% yield. In addition, the use of tandem affinity columns containing different protein binding sites allows the simultaneous purification of multiple DNA binding proteins from the same extract. This method provides a means for the purification of rare sequence-specific DNA binding proteins, such as Sp1 and CAAT-binding transcription factor.