Design of a zinc finger protein binding a sequence upstream of the A20 gene.
Design of a zinc finger protein binding a sequence upstream of the A20 gene.
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结合 A20 基因上游序列的锌指蛋白的设计
DOI：
10.1186/1472-6750-8-28
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发表时间：
2008-03-19
影响因子：
3.5
通讯作者：
Zhu C
中科院分区：
文献类型：
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作者：
Wei Y;Ying D;Hou C;Cui X;Zhu C
人工转录因子（ATF）由DNA结合和功能域组成。这些结构域可以融合在一起，以创建可以结合所选DNA序列的蛋白质。要构建有效的ATF，必须设计合适的DNA结合和功能域。 Cys2-His2锌指基序是构造序列特异性蛋白的理想结构支架。 A20是一种细胞质锌指蛋白，抑制核因子Kappa-B活性和肿瘤坏死因子（TNF）介导的程序性细胞死亡。已显示A20可防止TNF诱导的多种细胞类型的细胞毒性，包括成纤维细胞，B淋巴细胞，WEHI 164细胞，NIH 3T3细胞和内皮细胞。
为了设计结合A20基因启动子区域特定靶序列的锌指蛋白（ZFP）结构结构域，通过生物信息学方法分析了该启动子的结构和序列组成。 A20启动子中的目标序列已提交给Barbas实验室的在线ZF工具服务器Scripps Research Institute（TSRI），以获得特定的18 bp目标序列以及ZFP的氨基酸序列，该ZFP的氨基酸序列将结合到它。预测ZFP的序列表征和结构建模是通过生物信息学方法进行的。将这种人造ZFP的优化DNA序列重新组合到真核表达载体Pires2-EGFP中，以构建Pires2-EGFP/ZFP-FLAG重组剂，并通过RT-PCR，Western Pring和Emsa，Emsa和Emsa，EMSA，EMSA，EMSA，EMSA，EMSA，EMSA，ZFP的表达和生物学活性分析分别。 ZFP的设计成功并表现出生物学活性。
通过生物信息学方法设计特定的锌指蛋白是可行的。
Artificial transcription factors (ATFs) are composed of DNA-binding and functional domains. These domains can be fused together to create proteins that can bind a chosen DNA sequence. To construct a valid ATF, it is necessary to design suitable DNA-binding and functional domains. The Cys2-His2 zinc finger motif is the ideal structural scaffold on which to construct a sequence-specific protein. A20 is a cytoplasmic zinc finger protein that inhibits nuclear factor kappa-B activity and tumor necrosis factor (TNF)-mediated programmed cell death. A20 has been shown to prevent TNF-induced cytotoxicity in a variety of cell types including fibroblasts, B lymphocytes, WEHI 164 cells, NIH 3T3 cells and endothelial cells. In order to design a zinc finger protein (ZFP) structural domain that binds specific target sequences in the A20 gene promoter region, the structure and sequence composition of this promoter were analyzed by bioinformatics methods. The target sequences in the A20 promoter were submitted to the on-line ZF Tools server of the Barbas Laboratory, Scripps Research Institute (TSRI), to obtain a specific 18 bp target sequence and also the amino acid sequence of a ZFP that would bind to it. Sequence characterization and structural modeling of the predicted ZFP were performed by bioinformatics methods. The optimized DNA sequence of this artificial ZFP was recombined into the eukaryotic expression vector pIRES2-EGFP to construct pIRES2-EGFP/ZFP-flag recombinants, and the expression and biological activity of the ZFP were analyzed by RT-PCR, western blotting and EMSA, respectively. The ZFP was designed successfully and exhibited biological activity. It is feasible to design specific zinc finger proteins by bioinformatics methods.
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影响因子：
5.4
作者：
Eberhardy, SR;Goncalves, J;Barbas, CF
通讯作者：
Barbas, CF
影响因子：
56.9
作者：
Lee, EG;Boone, DL;Ma, A
通讯作者：
Ma, A
DOI：
10.1073/pnas.040552697
发表时间：
2000-02-15
影响因子：
11.1
作者：
Beerli, RR;Dreier, B;Barbas, CF
通讯作者：
Barbas, CF
影响因子：
5.6
作者：
Dreier, B;Segal, DJ;Barbas, CF
通讯作者：
Barbas, CF
影响因子：
3.7
作者：
通讯作者：
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