Expression of Zinc Finger Protein Zat12 from Arabidopsis thaliana in Escherichia coli

The C2H2 zinc finger protein ZAT12 has been classified as a plant core abiotic stress response gene in the early response to multiple stresses. ZAT12 links the iron deficiency and oxidative stress responses through the direct interaction with/and negative regulation of a central regulator FIT. For further research on the regulation of the ZAT12 protein in planta, a huge quantity of ZAT12 proteins is required to inject into mice for the generation of ZAT12 antiserum. In this study, the gene encoding the ZAT12 protein from Arabidopsis thaliana was cloned into the expression vector pETBlue-2 and then overexpressed in E. coli T7. A high expression level was indicated by SDS-PAGE. Immunoblot demonstrated successful expression using a bacterial expression system.

Recently, ZAT12 was identified in an attempt to find a link between iron deficiency responses and oxidative stress. In conditions where iron is poorly available, plants are able to induce a set of genes that enhance the efficiency of its uptake by the roots. A central regulator of this response in the model plant Arabidopsis (Arabidopsis thaliana) is the basic helix-loophelix transcription factor FIT whose activity is tightly regulated. Using a yeast two-hybrid assay and BiFC, the EAR motif was demonstrated to be necessary for the interaction between FIT and ZAT12 (Le et al., 2016). The expression of the FIT gene was upregulated in zat12 loss-offunction plants. In addition, these plants accumulated a higher amount of iron compared to the wild type. ZAT12-GFP fluorescence was detected in Arabidopsis roots, where it could be observed in the nuclei. This protein was elevated upon iron deficiency, H2O2, and MG132 treatments. ZAT12 is considered as a link between iron deficiency and oxidative stress responses through direct interaction with and negative regulation of FIT. These functions of the ZAT12 protein were only demonstrated using a ZAT12-GFP protein with a GFP antibody in immunoblot experiments (Le et al., 2016). To verify this native ZAT12 function, i.e to monitor Fe dependent expression and the regulation of ZAT12 protein in planta, it is necessary to generate an anti ZAT12 antibody. To do this, we have conducted a series of experiments including the cloning, transformation, heterologous expression, and purification of the recombinant protein. In this study, the ZAT12 recombinant protein was successfully expressed in E. coli.

Materials and methods
Strains, expression vector, and culture conditions for cloning E. coli Nova Blue cells were used as hosts for transformation. E. coli strains carrying the pETBlue-2 vector (Figure 1) were grown in Luria-Bertani medium supplemented with 100μg of tetracycline/mL when required at 37°C. The DNA fragments for subcloning were isolated from agarose gels and cloned into the pETBlue-2 vector using a Perfectly Blunt® Cloning Kit. (Novagen, Germany). Tuner (DE3) pLacI E. coli host strains that are compatible with the pET expression systems from Novagen were used for the expression of the ZAT12 protein. Other standard microbial and recombinant techniques used throughout this work were as described by Sambrook et al. (2001).

Generation of the ZAT12-His gene constructs using PCR
The coding sequence of ZAT12 from Arabidopsis thaliana was amplified by PCR using the primer combination 5' ZAT12_PET (5'-ATGGTTGCGATATCGGAGATCAA -3') ( Figure 2) and ZAT12_CT His 3' (5'-TCAAGAGGCCATACCGTGATGATGATGA TGATGAGAACCACGATAAACTGTTCT TCCAAGCTCCA -3') (Le et al., 2016). After checking the size of these fragments by agarose electrophoresis, the ZAT12-His fragments were purified and cloned into the EcoRV cloning site of the pETBlue-2 vector using the Perfectly Blunt® Cloning Kit.

Transformation
The recombinant plasmid was transformed into NovaBlue Singles™ Competent Cells (Novagen, USA). Identification of successful cloning events was performed by blue/white screening. The insertion of ZAT12-His into the pETBlue-2 vector leads to the disruption of the expression of the lacZ α-peptide, and thereby produces white colonies on plates supplemented with X-gal and IPTG (Isopropyl-β-D_thiogalactoside from Roth, Karlsruhe, Germany). Inversely, colonies containing the vector without insertion turn blue. After the initial selection of positive colonies, colony PCR and sequencing were performed for verification of positive recombinant plasmids. After that, the recombinant plasmid was transformed into Tuner (DE3) pLacI cells and the recombinant protein induction was performed according to the manufacturer's instructions (Novagen, USA).

Protein electrophoresis and Immuno Blot
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to determine the molecular mass of the protein using 10% gels (Laemmli et al., 1970). The bacterial extract containing the recombinant ZAT12 fusion protein was loaded on a preparatory gel and blotted to a nitrocellulose membrane. After Ponceau S staining, the membrane region containing the ZAT12 antigen was cut off as a strip. The membrane was blocked for 1 hour at room temperature with 1% bovine serum albumin (BSA) dissolved in PBS-T and subsequently probed with crude mouse antiserum at 4°C overnight. The unbound fraction was collected into a new tube. The membrane was washed 3 times with PBS-T and the bound antibodies were eluted two times with elution buffer (0.1 M glycine-HCl pH 2.7, 0.5 M NaCl). The eluted antibody fractions were immediately neutralized by adding 1/10 volume of neutralization buffer (1 M Tris-HCl pH 8.0, 1.5 M NaCl, 1 mM EDTA, 0.5% NaN3) and BSA was added at a 1 mg mL -1 final concentration (Novagen, according to manufacturer's manual).

Vietnam Journal of Agricultural Sciences
For the detection of the ZAT12-His protein, His antibodies and freshly purified undiluted anti-ZAT12 mouse antiserum were applied. These primary antibodies were detected with anti-mouse IgG conjugated with horseradish peroxidase (1:8000 dilution, Sigma-Aldrich, USA).
The ZAT12-His protein was detected by incubation with His rat antibody (1:1000 Roche, Germany) and a secondary antibody anti-rat IgG (whole molecule)-horse radish peroxidase conjugate (1:10000, Sigma-Aldrich, USA). Detection signals were developed using an

ZAT12 antibody preparation
Based on the predicted antigenic propensity scores, a peptide corresponding to the N-terminal of ZAT12 was chemically synthesized and conjugated with KLH (Bio Trend) and later injected into mice to obtain a polyclonal antiserum (this work was conducted by Prof. U. Müller, Zoology Department, Saarland University). In this study, the antiserum was tested for its specificity by detecting bacteria positively expressing ZAT12.

Plasmid construction and confirmation of cloned recombinant ZAT12 plasmid
To generate the recombinant plasmid, the full-length DNA sequence of ZAT12 was specifically amplified with the ZAT12-PET and ZAT12-CT-His primers, which produced an expected band of 538bp. Upon successful ligation and transformation, the obtained colonies were numbered and a colony PCR was performed to check the positive clones for the presence of the recombinant plasmid. In addition, sequencing of the selected recombinant plasmid was performed to confirm the proper orientation of the insert by ligation (Figure 3).
Resultant colonies were tested for the presence of the recombinant plasmid by colony PCR, and colonies were numbered as 1, 2, 3,…, 10. If the insert was in the correct orientation, the expected size of the PCR product for ZAT12 with the primer combination (ZAT12 5' and pETBlueDOWN, see Figure 2) was approximately 800bp (538bp of ZAT12 full plus 232bp from the pET Blue2 vector). Colony no. 8 of ZAT12 gave a PCR product at the expected size.

Expression of ZAT12 protein
After verifying the sequence, the plasmid was transformed into BL21 (DE3) cells of E. coli for expression. SDS-PAGE analysis detected the expected products from BL21 (DE3) cells induced with IPTG. The results are shown in Figure 4.
After colony PCR confirmation, the selected recombinant plasmid was transformed into the Tuner™ DE3 expression cells. Upon successful expression of the recombinant 18kDa ZAT12-His fusion protein at a small scale level, a large scale expression of ZAT12 protein was performed. at the 5'-ends of ZAT12 introduced via PCR. C. Colony-PCR of ZAT12-His colonies was performed on 10 colonies. Resultant colonies were numbered as 1, 2, 3,… ,10. Asterisks (*) indicate the ~800bp size ZAT12 fusion DNA band (from 538bp of ZAT12 full plus 232bp from the pET Blue2 vector). The only colony no. 8 gave a PCR product at the expected size, Figure 1A. M=ladder. Colony no. 8 was a positive colony.

Expression of Zinc Finger Protein Zat12 from Arabidopsis thaliana in Escherichia coli
Vietnam Journal of Agricultural Sciences

Specificity test of the ZAT12 protein
The heterologously expressed recombinant ZAT12 was purified for affinity and injected into mice. The immunization of mice and collection of antiserum were kindly performed by Prof. Uli Müller and Iris Fuchs, Department of Zoology, Saarland University. The obtained antiserum was checked for its specificity on E. coli expressing the ZAT12 fusion protein (Figure 5). We detected a single band on western blot that matched to the specifically expressed and desired ZAT12 protein so that we were able to conclude that the ZAT12 protein was expressed successfully in E. coli.

Discussion
The C2H2-type plant-specific zinc finger transcription factor family was defined by the presence of a conserved zinc finger domain, in which two cysteines and two histidines coordinate with a single zinc atom to form a finger construct consisting of a short beta-hairpin and an alpha helix. At5g59820 was identified among the genes belonging to this family and named ZINC FINGER OF ARABIDOPSIS THALIANA12 (ZAT12). Structural analysis of the ZAT12 protein showed that it constituted of 162 amino acids divided into two C2H2-type zinc finger domains with a 22-amino acid interfinger region, and a LDLSL core sequence of the EAR motif localized at the C terminus from amino acid 143 (Meissner & Michael, 1997;Englbrecht et al., 2004;Kagale et al., 2010). In plants, the ethylene-responsive element binding factor associated with the Amphiphilic Repression (EAR) motif is a transcriptional regulatory motif found as an active repressor in members of the ERF, C2H2, and AUX/IAA families, among others (Kagale et al., 2010). At5g59820 was identified to be a homolog of the Indica rice ZOS3-22 (Os03g0820400, LOC_Os03g6057, ZFP37). ZOS3-22 expression was also up-regulated in seedlings with a H2O2 treatment and in the shoots of plants exposed to Fe deficiency or combined Fe and P deficiency (de Abreu Neto & Frei, 2015).
The coding sequence of ZAT12 from Arabidopsis thaliana was amplified using touchdown PCR. The annealing temperature of 58ºC was suitable for amplification of the ZAT12 gene. Determination of the optimum annealing temperature for PCR is very important because total genomic DNA extracted from Arabidopsis thaliana was used as a template for ZAT12 amplified PCR (Rychlik et al., 1990). The fragment was a band with a size of 538bp, similar to the expected ZAT12 fragment size suggesting successful amplification of Zat12 from Arabidopsis thaliana genomic DNA using ZAT12_PET and ZAT12_CT His primers at the annealing temperature of 58°C.
The transformed Nova Blue bacterial cells were selected using 100 µg mL -1 tetracycline due to the presence of the tetracycline resistance gene in pETBlue-2. Colony PCR on the randomly selected bacterial colonies was conducted using the primers ZAT12 5' and pETBlue DOWN which binds to the vector downstream of the cloning site to identify transformed bacteria that carry recombinant pETBlue-2 with Zat12 inserted at the desired orientation. Also, the amplified fragment was approximately 800bp, consisting of 538bp of ZAT12 full and 272bp from the pET Blue2 vector. Lane 8 displayed a band with a size of approximately 800bp ( Figure  1C). Furthermore, lanes 1, 2, 3, 4, 5, 6, 7, 9, and 10 were similar in that they showed the absence of a band with a size smaller than 1kb. The results suggested that the colony coded 8 (lane 8) may carry the recombinant pETBlue-2 with Zat12 insert at the desired orientation. This assumption was tested through isolation of the plasmid from colony coded 8, which was followed by sequencing. The results agreed with the gene map published in the pETBlue-2 Cloning Kits User Manual. Hence, the results suggested that Zat12 was inserted into the cloning site of pETBlue-2 at the desired orientation. The converted sequence of ZAT12 showed the ZAT12 sequence-tagged 6x His had a band with a size of 1kb (refer to Figure 3). The results further confirmed that these plasmid DNA samples were pENTR™/D-TOPO® with Zat12 inserted in the cloning site at the desired orientation. A high expression level of the ZAT12 protein was indicated by SDS-PAGE and Immunoblot. Using His antibody and ZAT12 antiserum in Immuno Blot to determine the specificity of ZAT12 showed that ZAT12 was expressed successfully in E. coli.

Conclusions
Recombinant ZAT12 was expressed successfully in E.coli. This study helped accumulate enough ZAT12 recombinant protein for immunizing/injecting into mice in the following study of ZAT12 antibody generation.