A NONRADIOACTIVE ASSAY FOR THE DETECTION AND QUANTITATION OF A DNA-BINDING PROTEIN
A NONRADIOACTIVE ASSAY FOR THE DETECTION AND QUANTITATION OF A DNA-BINDING PROTEIN
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DOI：
10.1093/nar/22.18.3806
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发表时间：
1994-09-11
影响因子：
14.9
通讯作者：
CRAWFORD, L
中科院分区：
文献类型：
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作者：
HIBMA, MH;ELY, SJ;CRAWFORD, L
我们已经开发了一种替代方法来测定已知DNA结合蛋白的DNA结合活性。与标准凝胶阻滞测定法相比，该方法无放射性，定量，并在测定开始数小时内产生结果。利用酶联免疫吸附试验（ELISA），可以很容易地比较蛋白质制剂的DNA结合活性。虽然这种检测方法是用于检测活性人乳头瘤病毒16型（HPV 16） E2蛋白，但它可以很容易地适用于任何已知的DNA结合蛋白，其中抗体是可用的。hpv16e2的DNA结合序列先前有报道为5'-ACCGNNNNCGGT-3'(1), 5‘- accannnntggt -3’两个位置的单核苷酸变化显著降低了该蛋白与DNA的结合能力。采用常规的粘端方法合成了5′- a - ccgaaatcggttgaaccgaaaccggt -3′和5′- acca - ccgctggttgaaccaccgctggt -3′序列的正、反寡核苷酸。寡核苷酸在95℃冷却后退火，在15℃下用T4连接酶孵育过夜，苯酚提取和乙醇沉淀后，根据制造商的说明使用光探针（Vector Laboratories）进行生物素化。作为对照，一个不相关的寡核苷酸5‘- atataataatactaaactacaataat - tc -3’也被生物素化。将连接的寡核苷酸以lmg/ml的浓度重悬，并在20℃下保存直至需要。E2蛋白与谷胱甘肽- s -转移酶（GST）融合表达。通过聚合酶链反应从pBR-HPV16(2)中扩增出HPV16 E2开放阅读框，并克隆到pGEX （Pharmacia）。通过离心收集诱导表达GST-E2融合蛋白的细菌，并用超声波破坏。可溶性蛋白经谷胱甘肽树脂亲和纯化。该蛋白的GST部分在含有pGEX质粒的细菌中表达，没有插入物，并按照上述方法纯化。该蛋白在实验中作为对照
We have developed an alternative method for the assay of DNA binding activity of a known DNA binding protein. In contrast to standard gel retardation assays, this method is non-radioactive, quantitative and yields results within hours of commencement of the assay. Using an enzyme-linked immunosorbent assay (ELISA), the DNA binding activity of protein preparations can be easily compared. While this assay has been developed for the measurement of active human papillomavirus type 16 (HPV 16) E2 protein, it could be easily adapted for application to any known DNA binding protein for which antibodies are available. The DNA binding sequence of HPV16 E2 has been previously reported as being 5'-ACCGNNNNCGGT-3'(1) and single nucleotide changes at two positions to 5'-ACCANNNNTGGT-3'reduce the ability of the protein to bind to DNA significantly. Both forward and reverse oligonucleotides ofthe sequences 5'-A-CCGAAATCGGTTGAACCGAAACCGGT-3'and 5'-ACCA-CCGCTGGTTGAACCACCGCTGGT-3'were synthesised with sticky ends using conventional methodology. Oligonucleotides were annealed by cooling from 95 C and ligated by incubation with T4 ligase overnight at 15 C. Following phenol extraction and ethanol precipitation, oligonucleotides were biotinylated using photoprobe (Vector Laboratories) according to the manufacturer's instructions. For a control in the assay, an unrelated oligonucleotide 5'-ATATAATAATACTAAACTACAATAAT-TC-3'was also biotinylated. Ligated oligonucleotides were resuspended at a concentration of lmg/ml and stored at-20 C until required.The E2 protein used in the development of this assay was expressed as a fusion with glutathione-S-transferase (GST). The HPV16 E2 open reading frame was amplified by polymerase chain reaction from pBR-HPV16 (2) and cloned into pGEX (Pharmacia). Bacteria induced to express the GST-E2 fusion protein were harvested by centrifugation and disrupted by sonication. Soluble protein was affinity purified on glutathione resin. The GST portion of the protein was expressed in bacteria containing the pGEX plasmid with no insert and was purified as described above. This protein was used as a control in the