Protein Engineering Protocols - Mycobacteriology research center

Design of Zinc Finger Proteins 89proteins were constructed by linking two or three Sp1 zinc finger domains withthe Krüppel-type linker (see Notes 1 and 2).3.2.2. Construction of Genes for Sp1ZF6 and Sp1ZF9The pUC-Sp1(530-623), which codes for the Sp1 three-zinc finger region,was constructed as described previously (9).1. Prepare a synthetic oligonucleotide (84 bp) encoding the Krüppel-type linker(TGEKP) as BamHI/StyI fragment and insert this fragment into pUC-Sp1(530–623).2. Cut out the Eco47III fragment (264 bp) and insert into similarly digested pUC-Sp1(530-623). The plasmid is renamed as pUC-Sp1ZF6.3. Flank the middle Sp1 gene fragments for Sp1ZF9 with AgeI sites at the 5′ and 3′-endsby using the AgeI site primer pair, namely, 5′-ACCGGTGAAAAACCG-CATATTTGCCACATC-3′ for the coding strand, and 5′-CGGTTTTTCACCGGT-GTGGGTCTTGATATG-3′ for the noncoding strand.4. Ligate the resulting fragment modified with AgeI sites into the AgeI site of pUC-Sp1ZF6. The AgeI enzyme site between two and three Sp1 fragments encodes theamino acids TG, which are part of the TGEKP linker peptide.5. Confirm all sequences by DNA sequencing.6. Cut out the DNA fragments of Sp1ZF6 and Sp1ZF9 as a BamHI/EcoRI fragment andinsert into the similarly digested plasmid, pEV-3b (see Subheading 3.1.1.; ref. 10).3.3. DNA-Bending Zinc Finger ProteinsIn Subheadings 3.3.1. and 3.3.2., the design and the construction of DNAbendingzinc fingers are described.3.3.1. Strategy of Protein DesignBy linking two three-zinc fingers of Sp1, novel six-zinc finger proteins,including polyglycine linkers, Sp1ZF6(Gly) 4, Sp1ZF6(Gly) 7(Fig. 3), andSp1ZF6(Gly) 10, were created. Sp1ZF6(Gly) nwas constructed by exchangingthe TGEKP linker region of Sp1ZF6 with the (Gly) n(n = 4, 7, or 10) linkers(see Notes 3 and 4).3.3.2. Construction of Sp1ZF6(Gly) nSynthesized AflII/MfeI oligonucleotides containing the linker sequenceswere purchased from Amersham Biosciences. These oligonucleotides wereannealed and inserted into pUC-Sp1ZF6. These plasmids were renamed pUC-Sp1ZF6(Gly) n(n = 4, 7, or 10). The DNA fragments coding these proteins werecut out with BamHI and EcoRI, and inserted into the similarly digested plasmid,pEV-3b (see Subheading 3.1.1.; ref. 11).3.4. (His) 4-Type Zinc Finger ProteinThe procedures described in Subheadings 3.4.1. to 3.4.3. are the design strategy,peptide synthesis, and gene construction of (His) 4-type zinc fingers.