[Constructing and expression of three zinc-fingers peptide with specific DNA recognition property in Escherichia coli].
For investigating the DNA binding property of classical zinc finger protein Zif268, an in vivo transcription interference experiment was once utilized to develop a genetic selection assay. By screening a library in which the key amino acids of the third zinc finger from Zif268 were randomized, some single fingers with new binding specificity were obtained. In this study, by combining the single fingers, two three-finger peptides cDNA ZF123 and 2ZF123 were constructed by an over-lap PCR technique using the DNA binding domain of Zif268 as the template. After three times PCR, the products were inserted into pUC18 for cloning. The ZF123 and 2ZF123 cDNA were also inserted into pGEX-2T for expression in Escherichia coli after sequencing confirmation. The result showed that the three-finger peptides were expressed at a high level in E. coli JM109. The fusion protein GST-ZF123/2ZF123 have the relative molecular weight of 34.0 kD and consisted about 20% of the total soluble cell protein as detected by SDS-PAGE. After supersonic treatment, the soluble part of the bacterial extract was purified. After two additional thrombin cleavage and Sepharose 4B affinity purification steps, the free three-fingers peptide proteins were also obtained. The construction and obtaining of the three-fingers peptide cDNA and its products will facilitate the in vivo and in vitro DNA binding specificity study and the design of the hybrid transcription factors.
Zhang
S X
SX
Beijing Institute of Biotechnology, Beijing 100850, China.
Ma
Q J
QJ
Zhao
Z H
ZH
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D014157:Transcription Factors / Q000378:metabolism*; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D004247:DNA