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C-terminal in Sp1-like artificial zinc-finger proteins plays crucial roles in determining their DNA binding affinity.

Zhang B, Xiang S, Yin Y, Gu L, Deng D - BMC Biotechnol. (2013)

Bottom Line: Deletion of three amino acids in a consensus linker (TGEKP > TG) between finger-7 and the 6 × Histidine-tag in the C-terminal also dramatically abolished their binding affinity.In contrast, the replacement of the finger-3 in the N-terminal 3ZF domain did not affect their binding affinity, but decreased their binding stability.Altogether, the present study show that the C-terminal region may play crucial roles in determining the DNA binding affinity of Sp1-like artificial ZFPs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Division of Etiology, Peking University Cancer Hospital & Institute, Beijing 100142, China. dengdajun@bjmu.edu.cn.

ABSTRACT

Background: It is well known that the C-terminal zinc-finger-3 in transcription factor Sp1 contributes more than the N-terminal zinc-finger-1 in determining Sp1's DNA binding capacity. Sp1-like artificial poly-zinc-finger proteins (ZFPs) are powerful biotechnological tools for gene-specific recognization and manipulation. It is important to understand whether the C-terminal fingers in the Sp1-like artificial ZFPs remain crucial for their DNA binding ability. Recently, a set of p16 promoter-specific seven-ZFPs (7ZFPs) has been constructed to reactivate the expression of methylation-silenced p16. These 7ZFPs contain one N-terminal three-zinc-finger domain of Sp1 (3ZF), two Sp1-like two-zinc-finger domains derived from the Sp1 finger-2 and finger-3 (2ZF) in the middle and C-terminal regions.

Results: In the present study, sets of variants for several representative 7ZFPs with the p16-binding affinity were further constructed. This was accomplished through finger replacements and key amino acid mutations in the N-terminal fingers, C-terminal fingers, and linker peptide, respectively. Their p16-binding activity was analysed using gel mobility shift assays. Results showed that the motif replacement or a key amino acid mutation (S > R) at position +2 of the α-helix in the C-terminal 2ZF domain completely abolished their p16-binding affinity. Deletion of three amino acids in a consensus linker (TGEKP > TG) between finger-7 and the 6 × Histidine-tag in the C-terminal also dramatically abolished their binding affinity. In contrast, the replacement of the finger-3 in the N-terminal 3ZF domain did not affect their binding affinity, but decreased their binding stability.

Conclusions: Altogether, the present study show that the C-terminal region may play crucial roles in determining the DNA binding affinity of Sp1-like artificial ZFPs.

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Total protein and purified 7ZFP Active-H in different preparations from Rosetta DE3 bacteria transformed with the pET-28 7ZFP or empty control vectors. (a) Total crude proteins expressing 7ZFP (line-pointed band), Coomassie Blue staining. (b) The expression of 7ZFP Active-D was displayed in Western blot assay using antibody against 6 × Histidine-tag (Lane-1, total protein for pET-28a control vector; Lane-2, total protein for pET-28a-7ZFP vector; Lane-3, the sonicated supernatant for pET-28a-7ZFP vector; Lane-4, the sonicated precipitate for pET-28a-7ZFP vector; Lane-5, 7ZFP purified from the sonicated supernatant using Ni-NTA-Sepharose beads).
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Figure 3: Total protein and purified 7ZFP Active-H in different preparations from Rosetta DE3 bacteria transformed with the pET-28 7ZFP or empty control vectors. (a) Total crude proteins expressing 7ZFP (line-pointed band), Coomassie Blue staining. (b) The expression of 7ZFP Active-D was displayed in Western blot assay using antibody against 6 × Histidine-tag (Lane-1, total protein for pET-28a control vector; Lane-2, total protein for pET-28a-7ZFP vector; Lane-3, the sonicated supernatant for pET-28a-7ZFP vector; Lane-4, the sonicated precipitate for pET-28a-7ZFP vector; Lane-5, 7ZFP purified from the sonicated supernatant using Ni-NTA-Sepharose beads).

Mentions: To obtain soluble 7ZFPs, two distinct representative 7ZFPs [Active-H and Variant-F(f6) with the maximum number of amino acid differences] were inserted into six different kinds of expression vectors, including pET-28a, pET-30a, pET-32a, pMal-p2X, pGEX-4T-1, and pQE-Trisystem. Following transforming these vectors, the expression level and percentage of the soluble 7ZFPs in the corresponding bacteria were compared under various culture temperatures (10°C, 17°C, and 25°C) and IPTG induction times (12 hr and 24 hr). Results showed that soluble Active-H was most efficiently expressed in the pET-28a vector-transfected Rosetta (DE3) bacteria, after treatment of 0.1 mM of IPTG at 17°C for 24 hr (Figure 2). Most 7ZFPs expressed in the pET-28a vector were dissolved in the sonicated supernatants (about 25 kD). Similar results were obtained for Variant-F(f6) (data not shown). Therefore, the pET-28a vector was used to express the soluble 7ZFPs for further experiments. After purification using Ni-NTA-Sepharose beads, the soluble 7ZFPs were detected as a marked specific band and confirmed by Western blot (Figure 3). Such a band was not observed in the uninduced or pET-28a transformed-control bacteria.


C-terminal in Sp1-like artificial zinc-finger proteins plays crucial roles in determining their DNA binding affinity.

Zhang B, Xiang S, Yin Y, Gu L, Deng D - BMC Biotechnol. (2013)

Total protein and purified 7ZFP Active-H in different preparations from Rosetta DE3 bacteria transformed with the pET-28 7ZFP or empty control vectors. (a) Total crude proteins expressing 7ZFP (line-pointed band), Coomassie Blue staining. (b) The expression of 7ZFP Active-D was displayed in Western blot assay using antibody against 6 × Histidine-tag (Lane-1, total protein for pET-28a control vector; Lane-2, total protein for pET-28a-7ZFP vector; Lane-3, the sonicated supernatant for pET-28a-7ZFP vector; Lane-4, the sonicated precipitate for pET-28a-7ZFP vector; Lane-5, 7ZFP purified from the sonicated supernatant using Ni-NTA-Sepharose beads).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4219604&req=5

Figure 3: Total protein and purified 7ZFP Active-H in different preparations from Rosetta DE3 bacteria transformed with the pET-28 7ZFP or empty control vectors. (a) Total crude proteins expressing 7ZFP (line-pointed band), Coomassie Blue staining. (b) The expression of 7ZFP Active-D was displayed in Western blot assay using antibody against 6 × Histidine-tag (Lane-1, total protein for pET-28a control vector; Lane-2, total protein for pET-28a-7ZFP vector; Lane-3, the sonicated supernatant for pET-28a-7ZFP vector; Lane-4, the sonicated precipitate for pET-28a-7ZFP vector; Lane-5, 7ZFP purified from the sonicated supernatant using Ni-NTA-Sepharose beads).
Mentions: To obtain soluble 7ZFPs, two distinct representative 7ZFPs [Active-H and Variant-F(f6) with the maximum number of amino acid differences] were inserted into six different kinds of expression vectors, including pET-28a, pET-30a, pET-32a, pMal-p2X, pGEX-4T-1, and pQE-Trisystem. Following transforming these vectors, the expression level and percentage of the soluble 7ZFPs in the corresponding bacteria were compared under various culture temperatures (10°C, 17°C, and 25°C) and IPTG induction times (12 hr and 24 hr). Results showed that soluble Active-H was most efficiently expressed in the pET-28a vector-transfected Rosetta (DE3) bacteria, after treatment of 0.1 mM of IPTG at 17°C for 24 hr (Figure 2). Most 7ZFPs expressed in the pET-28a vector were dissolved in the sonicated supernatants (about 25 kD). Similar results were obtained for Variant-F(f6) (data not shown). Therefore, the pET-28a vector was used to express the soluble 7ZFPs for further experiments. After purification using Ni-NTA-Sepharose beads, the soluble 7ZFPs were detected as a marked specific band and confirmed by Western blot (Figure 3). Such a band was not observed in the uninduced or pET-28a transformed-control bacteria.

Bottom Line: Deletion of three amino acids in a consensus linker (TGEKP > TG) between finger-7 and the 6 × Histidine-tag in the C-terminal also dramatically abolished their binding affinity.In contrast, the replacement of the finger-3 in the N-terminal 3ZF domain did not affect their binding affinity, but decreased their binding stability.Altogether, the present study show that the C-terminal region may play crucial roles in determining the DNA binding affinity of Sp1-like artificial ZFPs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Division of Etiology, Peking University Cancer Hospital & Institute, Beijing 100142, China. dengdajun@bjmu.edu.cn.

ABSTRACT

Background: It is well known that the C-terminal zinc-finger-3 in transcription factor Sp1 contributes more than the N-terminal zinc-finger-1 in determining Sp1's DNA binding capacity. Sp1-like artificial poly-zinc-finger proteins (ZFPs) are powerful biotechnological tools for gene-specific recognization and manipulation. It is important to understand whether the C-terminal fingers in the Sp1-like artificial ZFPs remain crucial for their DNA binding ability. Recently, a set of p16 promoter-specific seven-ZFPs (7ZFPs) has been constructed to reactivate the expression of methylation-silenced p16. These 7ZFPs contain one N-terminal three-zinc-finger domain of Sp1 (3ZF), two Sp1-like two-zinc-finger domains derived from the Sp1 finger-2 and finger-3 (2ZF) in the middle and C-terminal regions.

Results: In the present study, sets of variants for several representative 7ZFPs with the p16-binding affinity were further constructed. This was accomplished through finger replacements and key amino acid mutations in the N-terminal fingers, C-terminal fingers, and linker peptide, respectively. Their p16-binding activity was analysed using gel mobility shift assays. Results showed that the motif replacement or a key amino acid mutation (S > R) at position +2 of the α-helix in the C-terminal 2ZF domain completely abolished their p16-binding affinity. Deletion of three amino acids in a consensus linker (TGEKP > TG) between finger-7 and the 6 × Histidine-tag in the C-terminal also dramatically abolished their binding affinity. In contrast, the replacement of the finger-3 in the N-terminal 3ZF domain did not affect their binding affinity, but decreased their binding stability.

Conclusions: Altogether, the present study show that the C-terminal region may play crucial roles in determining the DNA binding affinity of Sp1-like artificial ZFPs.

Show MeSH
Related in: MedlinePlus