Design of a zinc finger protein binding a sequence upstream of the A20 gene.
BACKGROUND
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.


RESULTS
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.


CONCLUSIONS
It is feasible to design specific zinc finger proteins by bioinformatics methods.
Wei
Yong
Y
The Key Laboratory of Biomechanics and Tissue Engineering of Chongqing Municipality, Department of Anatomy, Third Military Medical University, Chongqing, 400038, China. yongwei70@gmail.com
Ying
Dajun
D
Hou
Chunli
C
Cui
Xiaoping
X
Zhu
Chuhong
C
D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D014157:Transcription Factors / Q000235:genetics*; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*; D016335:Zinc Fingers / Q000502:physiology*
D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; C076529:TNFAIP3 protein, human; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3