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

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.In nature, gene expression is regulated at the transcriptional level primarily by transcription factors that bind to DNA. Many of these transcription factors consist of two essential yet separable modules: a DNA-binding domain and a functional domain [1-3]. Artificial transcription factors (ATFs) are composed of DNA-binding and functional domains [4-6], which can be fused together to create proteins that bind a chosen DNA sequence and regulate expression of a specific gene in vivo [1,2,4,7,8]. Construction of ATFs in vitro includes construction of DNA-binding and function domains by various methods. It is very important to design a DNA-binding domain that recognizes a specific DNA sequence. Recently there has been a great deal of progress in the development of modular protein domains that recognize specific DNA triplets. The Cys2-His2 zinc finger motif is the ideal structural scaffold on which a sequence-specific protein may be constructed [7,9]. DNA st