Among DNA-binding modulators, (Cys)2(His)2-type zinc-finger modulators have a large potential for manipulation. Zinc finger modulators provide an attractive framework for the design of novel DNA binding proteins. In particular, zinc finger modulators provide an attractive framework for generating new artificial zinc finger proteins with novel DNA binding properties, such as long DNA strand recognition, DNA bending, and AT-rich sequence recognition. The source of this experiment is "A Guide to Modern Protein Engineering Experiments" [German] K.M. Arndt, K.M. Miller, eds.
Operation method
Design and synthesis of artificial zinc finger proteins
Materials and Instruments
Oligonucleotide primers Move The methods described in this section probably include: For more product details, please visit Aladdin Scientific website.
Ampicillin Phosphate-buffered saline Tris-hydrogen chloride SDS-PAGE
DNA sequencer Chromatography equipment
( 1 ) Expression and purification of designed zinc finger proteins.
( 2 ) Artificial zinc finger design techniques.
( 3 ) Steps of artificial zinc finger construction.
( 4 ) Confirmation of zinc finger structure and metal coordination.
3.1 Expression and purification of artificial zinc finger protein
The methods designed for the expression and purification of artificial zinc finger proteins are described in sections 3.1.1 and 3.1.6 and include:
( 1 ) Construction of expression vector pEV-3b;
( 2 ) Expression of the protein in soluble and insoluble forms;
( 3 ) Purification of the protein by cation exchange and gel filtration chromatography;
( 4 ) Refolding of inactivated structures;
( 5 ) Confirmation of zinc finger structure and metal coordination.
3.1.1 pEV-3b Expression Vector
The pEV-3b expression vector is based on the pET3b expression vector (Novagen; Figure 5.1 ). This vector was modified to generate useful polyclonal sites for the study of zinc finger genes.
( 1 ) Excision of EcoRI/Hind lll degraded fragments from pET3b.
( 2 ) To excise the EcoRI and sites, annealed oligonucleotides 5'-AATTGTCATGTTTGAC-3, and 5,- AGCTGTCAAACATGAC-3, were inserted into the EcoRI/Hind lll degraded pET3b. The resulting plasmid is labeled pET3b'.
( 3 ) Preparation of a double-stranded oligonucleotide including the restriction enzymes AflII, Bam HI, EcoRI, Hind lll, and S mal cleavage sites. The sequences of these two strands are 5'-TATGGATCCCGGGGAATTCAAGCTTAAGC-3' and 5'-TCAGCTTAAGCTTGAATTCCCGGGGATCCA-3'.
( 4 ) Degraded with Nde l and and inserted this fragment into pET3b' to obtain plasmid pEV-3b.
The obtained plasmid pEV-3b was used to express the designed zinc finger protein. By inserting the zinc finger gene as a Bam HI/EcoRI fragment into the similarly degraded pEV-3b, an artificial zinc finger expression vector was constructed in a single step. 
3.1.2 Expression of the designed artificial zinc finger protein
( 1 ) Transform the pEV-3b plasmid with the designed zinc finger protein gene into E. coli strain BL21 ( DE3 ) pLysS.
( 2 ) Cultivate the cells at 37°C in Luria-Bertani medium.
( 3 ) Add 1 mmol/L IPTG when the optical density at 600 nm is 0.6.
( 4 ) The culture medium was incubated at 20°C for 8~12 h. This temperature is important for the expression of proteins in the solubilized state.
3.1.3 Purification of designed artificial zinc finger protein
The purification step was done at 4°C.
( 1 ) Harvest E. coli cells, resuspend, and lysed in PBS.
( 2 ) After centrifugation, the supernatant with solubilized zinc finger proteins was purified by cation exchange chromatography (High S and UNO S-1; Bio- Rad) and gel filtration with Tris-hydrogen chloride buffer (Superdex 75; Amersham Biosciences).
( 3 ) Protein purity was confirmed by SDS-PAGE.
For metal substitution experiments, the insoluble form of zinc finger protein was also purified from E. coli cell pellets after centrifugation:
( 1 ) Cell pellets were lysed in PBS containing 8 mmol/L urea and 10 mmol/L chelating agent (EDTA or 1,10-o-diazepine).
( 2 ) Purify in the same steps as described in step (2) of subsection 3.1.3.
( 3 ) Heat the purified proteins at 65°C for 30 min and refold by gradual cooling in 10 mmol/L Tris buffer containing 125 μmol/L zinc chloride, nickel nitrate, cadmium chloride, cobalt nitrate, or copper sulfate.
3.1.4 CD measurement
CD spectra of zinc finger proteins are recorded with a Jasco J-720 spectropolarimeter at 20°C using Tris-HCl buffer pH 8.0 containing 50 mmol/L NaCl. The container must have a lid, 1 mm optical range, and nitrogen atmosphere. All spectra were averaged over 8 to 16 scans. The baselines of the spectra were calibrated and noise reduced using Jasco software.
3.1.5 UV-VIS Absorption Spectra
UV-VIS absorption spectra were obtained with a Beckman Coulter DU7400 diode array spectrometer at 20°C using 10 mmol/L Tris-HCl buffer containing 50 mmol/L NaCl at pH 7.5. The container must have a lid and a 1 cm light path. The vessel must have a lid and a 1 cm optical range.Co (II) - substituted zinc finger complexes were obtained by titration with cobalt chloride. Peptides were saturated with Co (I I ) under arbitrary conditions. All spectra are expressed as e = A / ( l/c), where e is the extinction coefficient {/ [ (mol/L ) /cm]}, l is the optical range of the container (cm), and c is the peptide concentration (mol/L ).
3.1.6 NMR experiment
Complexes of single finger structural domains and Zn (II) were prepared in 90% H2O/10%D2O and D2O ( 25 mmol/L Tris-d11, pH 5.7) in the presence of 1.5 mol of Zn ( II) ions at 5 mmol/L concentration. All NMR spectra were recorded on a JOEL Lambda-600 spectrometer.
( 1 ) Nuclear Overhauser Enhanced Spectroscopy (NOESY) data were acquired with selective water presaturation (alternating delays and feeds to tailor the excitation pulse; a water-peak signal suppression technique abbreviated as DANTE; mutation was taken to be nutation ---- ), followed by temperature setting at 303 K and mixing times of 100 ms, 200 ms, and 1 hr for each. The temperature was then set at 303 K, each using a standard NOESY pulse sequence with mixing times of 100 ms, 200 ms, and 300 ms, respectively.
( 2 ) Total correlation spectra were acquired with 80 ms MLEV-17 spin-lock duration, and a gradient-trimmed pulse at 303 K was used to suppress the water signal.
Typical acquisition conditions for the spectra were 24 scans per t1 value, for a total of 1024 t1 values, and 2048 complex points in the direct dimension. The free induction decay in both dimensions was multiplied by a phase-shifted sinusoidal bell-shaped limiting function, zero-padded, and Fourier transformed to a 2048X2048 matrix. Sequence resonance identifications were determined using standard total correlation spectra and the NOESY process [8].
3.2 6 and 9 zinc finger proteins
This section describes the design strategy and construction of multiple zinc finger proteins required for the recognition of long DNA sequences.
3.2.1 Protein design strategy
The novel 6 and 9 zinc finger proteins (SplZF6 and SplZF9 ) were created from the 3-zinc finger motif of the transcription factor Sp1 (Figure 5.2 ). These proteins were constructed by linking 2 or 3 Spl zinc finger domains with a Kriippel-type linker (see Note 1 and Note 2). 
3.2.2 Construction of the Sp1ZF6 and Sp1ZF9 Genes
The gene encoding the 3 zinc finger region of Sp1 [pUC-Spl ( 530~623)] was constructed as previously described [9].
( 1 ) An oligonucleotide (84 bp) encoding a Kriippel-type linker (TGEKP) was synthesized as a BamHI/Sty I fragment and inserted into pUC-Spl (530~623).
( 2 ) A fragment of Eco47 III (264 bp) was cut out and inserted into the simply degraded pUC-Sp1 (530~623). The altered plasmid was renamed pUC-Sp1ZF6.
( 3 ) The two AgeI sites were ligated to the 5' and 3' ends of the intermediate gene encoding SplZF9 using Agel primer pairs, i.e., 5'-ACCGGTGAAAAACCGCATATATTTGCCACATAC-3' as the coding strand and 5'-CGGTTTTTCACCGGTGTGGGTCTTGATATG-3' as the non-coding strand, respectively. end and 3' end of the intermediate gene encoding SplZF9, respectively.
( 4 ) Attach the resulting Agel-modified fragment to the AgeI position of pUC-SplZF6. The AgeI cleavage site between 2 or 3 Spl fragments encodes the amino acid TG, which is part of the linker peptide TGEKP.
( 5 ) Confirm all sequences by DNA sequencing.
( 6 ) Cut the DNA fragments of SplZF6 and SplZF9 as BamHI/Styl fragments and insert them into the briefly degraded plasmid pEV-3b (see 3.1.1; Ref. [10] ).
3.3 DNA Bending Zinc Finger Proteins
Sections 3.3.1 and 5.3.3.2 describe the design and construction of DNA bent zinc fingers.
3.3.1 Protein Design Procedure
A new 6-zinc finger protein was constructed by ligating the two 3-zinc fingers of SP1, including the polyglycine linker, SplZF6 (Gly)4, SplZF6 (Gly)7 (Fig. 5.3), and SplZF6 (Gly)1. Exchanging the TGEKP and (Gly)n (n = 4, 7, or 10 ) linkers of SplZF6, it was possible to construct SplZF6 ( Gly)n (see Note 3 and Note 4). 
3.2 Construction of SplZF6 (Gly )n
Synthetic oligonucleotides A flll/Mfel containing the linker sequence were purchased from Amer sham Biosciences. After annealing, these oligonucleotide fragments were inserted into the pUC-Sp1ZF6 vector. The resulting plasmid was renamed Sp1ZF6 (Gly )n ( n = 4 , 7 or 10). These protein-encoding DNA fragments were cut down at the Bam HI and Eco RI sites and inserted into a plasmid, pEV-3b, that was similarly cleaved (see 5. 3.1 .1; ref [ 11 ]).
3.4 (His )4-type zinc finger proteins
Sections 3.4.1 and 3.4.2 describe design techniques, peptide synthesis, and gene construction for (His )4-type zinc finger proteins.
3.4.1 Protein design strategy A new (His )4-type zinc finger protein was created by mutating CysHis on the 3 (Cys )2 (His )2-type zinc finger protein Sp1 (Figure 5.4). In essence, there are (Cys )2 (His )2 -, (Cys )3 (His) -, (Cys )4-, and (Cys )6 - zinc finger proteins (see Note 5). 
3.4.2 Peptide synthesis of (His )4-type zinc finger domains
Synthesis of a (His )4 mutant peptide (H4Sp1f2 ) of the middle finger of the 3 zinc finger Sp1 was synthesized on Rink amino resin using 9-fluorenylmethoxycarbonyl solid phase. The peptide chains were synthesized on a Shimadzu PSSM-8 synthesizer using the standard process of benzotriazol-1-yl oxo-3-tetrahydropyrrolidophosphate, hexafluorophosphoric acid, (PyBOP)-1-hydroxybenzotriazole, and (HOBO-N-methylmorpholine, (NMM ) coupled system. The protected H4Sp1f2 peptide resin was treated with trifluoroacetic acid and ethanol ( 95 : 5 ) for 2 h at room temperature, followed by purification by high performance liquid chromatography on a μBondesphere4C4-300 (19 X 50 mm) column. The reliability of the products was verified by time-of-flight mass spectrometry (Kratos Kompact MALDI4 ).
3.4.3 Construction of H4Sp1
The (His )4 mutant H4Sp1 was constructed by mutating the 6 cysteines in the 3 zinc finger domain of Sp1 to histidine. The DNA fragment encoding (His )4 was obtained from pUC-Sp1 (530-623) by point mutation-based polymerase chain reaction (PCR ) and verified by GeneRapidDNA (Amersham Biosciences ) sequencer. The amplified fragments were cut with BamHI and Eco RI and inserted into simply degraded pEY-3b (see 3.1.1; ref. [12]).
3.5 AT recognition of zinc finger proteins
The design and construction of AT-recognizing zinc fingers are described in sections 3.5.1 and 3.5.2, respectively.
3.5.1 Protein design strategy
The 3 zinc fingers in the (Cys )2 (His )2-type zinc finger protein Sp1 bind to GC-rich sequences, while the 3 zinc fingers (4-6 fingers) in the 6 (Cys )2 (His )2-type zinc finger protein CF2- II bind to AT-rich sequences. New zinc finger proteins that bind to AT-rich sequences are synthesized by exchanging the helices of these two types of zinc finger proteins (Figure 5.5). 
3.5.2 Construction of Sp1HM
Oligonucleotides encoding the α helix of CF2- II were prepared. PCR-based point mutations were retrograded using pUC-Sp1 ( 530~623 ) as a template. PCR-amplified mutant fragments were degraded with Bam HI and Eco RI and inserted into a simply degraded pEV-3b plasmid (see 3.1.1; ref. [13]).