| Budget Amount *help |
¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000)
Fiscal Year 2010: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
Fiscal Year 2009: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2008: ¥2,470,000 (Direct Cost: ¥1,900,000、Indirect Cost: ¥570,000)
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| Research Abstract |
Zinc finger protein is naturally occurring DNA-binding protein found in many transcription factors. Zinc finger protein has two precious molecular recognition abilities ; (1) metal ion binding and (2) DNA-binding abilities with high affinity and selectivity. Because of these characteristic, zinc finger protein is promising framework for designing artificial zinc finger proteins. In this project, I tried to introduce the catalytic activities, hydrolytic, nuclease and peptidase activities, to Sp1 zinc finger protein itself by redesign of metal binding site. At first, three His4-type zinc finger domains (FxH4, x=1,2 and 3) were created by mutation of two Cys residues to His residues for each three zinc finger domain of Sp1. All His4-type zinc finger peptides possess the high hydrolytic activities for BNPP having phosphate ester bond. Especially, F1H4 has much higher hydrolytic activity for phosphate ester compare to that of F2H4 and F3H4. Next, new types of fusionl zinc finger nuclease, ZWH4 and Sp1fxH4Sp1, were prepared. ZWH4 was created by connecting F2H4 domain to c-terminus of Sp1 zinc finger protein. ZWH4 could efficiently cleavage plasmid pUC19 DNA containing target sequence. In addition, ZWH4 showed also peptidase activity. In order to increase the binding affinity for target DNA sequence and the catalytic activity of Hie4-type zinc finger protein, Sp1FxH4Sp1 (x=1 and 2), 7-finger proteins, were created by combine wild-type Sp1 to both N- and C-terminus of FxH4 zinc finger domain. Sp1FxH4Sp1 can produce as His-tag fusion protein using E coli protein expression systems. In gel mobility shift assay, Sp1FxH4Sp1 could bind to target DNA sequence. Moreover, in preliminary DNA cutting experiment, Sp1F2H4Sp1 showed week hydrolytic activity for plasmid DNA.
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