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

, 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). 
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 full DNA 
 5’ ZAT12_PET 
pETBlueDOWN 
770 
bp pETBlue-2 vector 
Le Thi Tuyet Cham et al. (2019) 
https://vjas.vnua.edu.vn/ 507 
enhanced chemiluminescence detection kit 
(Biorad, USA) according to the manufacturer’s 
protocol. 
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. 
Results 
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. 
Figure 3. Preparation of the ZAT12 encoding gene. A. The DNA fragment of ZAT12-His using PCR. B. Amino acid changes 
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 
508 Vietnam Journal of Agricultural Sciences 
Figure 4. SDS-PAGE analysis of the heterologously expressed recombinant ZAT12 fusion protein in E. coli. ZAT12 fusion 
protein expression in Tuner™ DE3 cells accumulated as insoluble proteins (inclusion bodies) and soluble fraction. Asterisks 
(*) indicate the position of ~18kDa ZAT12 fusion protein band (from ZAT12 full gene plus 6 His tags). 
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 inter-
finger 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).
kDa 
Soluble 
fraction 
Insoluble 
fraction 
* * 
Le Thi Tuyet Cham et al. (2019) 
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Figure 5. Specificity of the ZAT12 protein. A. The last lane from the right-hand side was loaded with ZAT12 induced 
bacterial culture and probed/incubated with anti-His antibodies (A), and with anti-ZAT12 unpurified antiserum (B). The 
triangle ( ) indicates the position of the ~18kDa ZAT12 fusion protein band (from ZAT12 full gene plus 6 His tags). 
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 touch-
down 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 
Expression of Zinc Finger Protein Zat12 from Arabidopsis thaliana in Escherichia coli 
510 Vietnam Journal of Agricultural Sciences 
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. 
Acknowledgements 
The author would like to convey his her deep 
thanks to the Laboratory of Botany, Faculty of 
Biosciences, University, Saarbrueken, Germany 
for their kind hospitality and support in 
conducting this study. A special thanks to 
Professor Dr. Uli Mueller, Department of 
Zoology, Saarland University, for his kind help 
in producing anti-ZAT12 antiserum. 
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