Gene Therapy and Molecular Biology Vol 15, page 186
Gene Ther Mol Biol Vol 15, 186-193, 2013
Prokaryotic expression，purification of
Mycobacterium tuberculosis PZA-resistant
protein
Research Article
Jie Shi1*, Baolin Song2*, Hui Li1, Xiao-guang Ma 1, Yu-ling Zhao1, Guo-rui
Yan1, Jin Xing1
1 Henan Province Center for Disease Control and Prevention, Zheng zhou, 450016 China
2 Henan Vocantional and Technical College of Communications, Zhengzhou 450000, China,
* These authors contributed equally to this study
*Correspondence: Jie Shi Address: No.105 ,NongYe Road, Zheng zhou ,Henan, China; Tel: 0371-68089289;
Email:[email protected]
Keywords: Mycobacterium tuberculosis; pncA gene; pyrazinamide-resistance; pyrazinamidase; prokaryotic
expression
Received: 23 October 2013; Revised: 20 December 2013
Accepted: 26 December 2013; electronically published: 31 December 2013
Summary
Objective: To construct His-tag prokaryotic expression plasmid of the pncA gene of
Mycobacterium tuberculosis, and to express the fusion proteins efficiently in Escherichia coli
BL21. Methods: The pncA gene was amplified by PCR with specific primers from genomic
DNA of M.tuberculosis H37Rv strain, and was cloned into pET28a expression vector. E.coli
BL21 strain was transformed with the recombinant vector that conformed by sequencing
and induced to express recombinant proteins. The relative molecular size of the proteins was
analyzed by SDS-PAGE and immunoblot. Results: The length of PCR products pncA was
561 bp, and the sequence was identical to genomic DNA. The recombinant expressive vector
pET28a-pncA was constructed. The E.coli BL21 strains with recombinant vector showed
high level of pyrazinamidase fusion protein expressions after IPTG induction. Conclusion:
The pncA gene prokaryotic expression plasmid was constructed, and the pyrazinamidase
fusion protein was efficiently expressed after induction. The expression of the recombinant
pyrazinamidase protein lays a basis for further study on pyrazinamide-resistance of
M.tuberculosis.
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Shi et al: Prokaryotic expression, purification of Mycobacterium tuberculosis PZA-resistant protein
I. Introduction:
II. Materials and Methods:
China is ranked second among the 22
high-tuberculosis
(TB)-burden
countries
designated by the World Health Organization
(WHO), with an incidence rate of 367 cases
per 100,000 citizens in 2006. Tuberculosis
(TB) remains a major public health concern in
China, causing 150,000 deaths per year and
making China second only to India in TB
mortality(2008). A significant challenge for
TB control in China is the rapid dissemination
and high prevalence of drug-resistant
Mycobacterium tuberculosis(Wright, Zignol et
al. 2009).
Pyrazinamide （ PZA ） is
the
antituberculous drug of choice in modern
short-course regiment with isoniazid, rifampin,
and ethambutol (Zimic, Sheen et al. 2010).
PZA appear to kill semidormant tubercle
bacilli that persist in a low-pH environment
and are unaffected by any other
antiuberculous drug (Vargas, Rivas et al. 2003;
Singh, Wesley et al. 2007; Zimic, Fuentes et al.
2012) and allows shortening of the treatment
period from 12 to 18 months to 6 months
when combined with isoniazid and rifampin.
The exact mode of action of PZA still has not
been determined, but it is thought that
bacterial pyrazinamidase (PZase) converts
PZA to toxic pyrazononic acid, which
mediates the direct killing effect (Zimhony,
Cox et al. 2000; Zimic, Loli et al. 2012).
Previous reports have well characterized the
mutations of the pncA gene in PZA-resistant
MTB isolates (Pandey, Newton et al. 2009;
Chiu, Huang et al. 2011; Kim, Kwak et al.
2012; Lee, Yun et al. 2012). Therefore,
mutation in pncA is a majar mechanism of
pyrazinamide resistance in Mycobacterium
tuberculosis. In this study we first cloned
pncA prokaryotic expression vector, and
expressed
histidine-tagged
recombinant
protein in E.coil and purifed this protein using
affinity chromatography.
A. Strains and Plasmids.
The standard Mycobacterium tuberculosis strain,
H37Rv, was provide by the National Tuberculosis
Reference Laboratory of the Chinese Center for
Disease Control and Prevention, Beijing, China.
Expression Vectors pET-28a, and strains E. coli
Top10 and BL21(DE3) were saved in our lab. All
enzymes, agarose gel DNA fragment purification
kit purchased from TAKARA Co.(Kyoto, Japan),
other biochemical reagents were obtained from
Shenggong Co.(Shanghai, China). Ni Sepharose
for purification of His-tag protein was obtained
from Amersham Co.( Buckinghamshire,UK).
B. Construction of His-fusion Protein
Expression Vector.
M.tuberculosis was grown in neural LowensteinJensen medim for 2 to 4 weeks. Genomic DNA for
PCR was isolated as follows. Scraping the
bacterial cultures to 500 μl TE buffer, the bacterial
cells were killed by incubating them at 85℃ for 30
min. Genomic DNA was isolated from
M.tuberculosis as previous description(Scorpio,
Lindholm-Levy et al. 1997).
Two primers were designed ， based on M.
tuberculosis sequences retrieved from Genbank to
amplify approximately 600 bp of the PZase gene
(pncA) ORF region. Oligonucleotides were
designed with a BamHⅠ restriction site within the
5’ forward primer and an EcoRⅠ restriction site
within the 3’reverse primer. The forward primer
was
GGAT
CCATGC
GGGCGTTGAT
CATCGTCGAC, the reverse primer was GGCG
GAATTC TCAGGAGCTG CAAACCAACT CGG.
The ORF carrying pncA gene was PCR amplified
from a genomic DNA of M.tuberculosis strain
H37Rv. The amplified pncA gene was cloned in
the pET28a expression vector (Novagen). The
integrity of the plasmid clone was confirmed by
DNA sequencing.
C. Protein Expression and Purification.
For analytical protein expressions, cultures were
grown to OD600= 0.6-0.8 in LB medium containing
100g/mL ampicillin. Expression of the PZase was
induced by the addition of IPTG to a final
concentration of 1 mM. After 4-6 h, 1 mL of cells
was harvested and lysed in 0.2mL of lysis
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Gene Therapy and Molecular Biology Vol 15, page 188
A 50 bp DNA ladder was used for size
control of amplified fragments. There is an
exceptional bright DNA band of about 600bp
in size, which consistent with the size of
pncA gene.
buffer(containing 150mM NaCl，20mM Tris-HCl
(pH7.4)，5mM MgCl2，1%Triton X-100，1mM
DTT，1mM PMSF). Expressions were analyzed
by PAGE using 10% polyacrylamide gels. For
large-scale expressions, cultures were grown to
OD600 = 0.6-0.8 in 20mL of LB medium
containing 100mg/mL ampicillin, and the PZase
protein expression was induced by the addition of
IPTG to a final concentration of 1 mM. After 6h,
the cells were harvested by centrifugation and
resuspended in 20 ml PBS buffer containing
complete protease inhibitor cocktail. Cells were
then sonicated on ice for 2 min, and the
supernatant was incubated overnight with 1 ml Ni
Sepharose 6 Fast Flow at 4 ℃. The resin bound to
protein was packed into a column and washed with
five bed volumes of binding buffer(20 mM sodium
phosphate, 0.5 M NaCl, 5 mM imidazole, pH 7.4).
Protein was eluted with elution buffer (20 mM
sodium phosphate, 0.5 M NaCl, 0.5 M imidazole,
pH 7.4). Fractions were analyzed by 10% SDSPAGE gel. Fractions containing fusion protein
were pooled and stored at −70℃.
Figure 1: Electrophoretic result of pncA gene
after PCR.
D. Immunoblot Analysis of Purified
Proteins.
The prokaryotic expression plasmid
pET28a and pncA gene amplified by PCR
were digested by BamHⅠand EcoRⅠ,
respectively. The recycle target segment was
reconstructed and its
product
was
transformed in E.coil Top10. Colony PCR
was performed to rapidly screen E.coil
recombinant clones and to verify the insert
size of destination clones. Five bacterial
clones were randomly picked and identified.
For pncA constructs, the size of PCR
amplicons should be the length of
approximately 600 bp (Figure 2). Three
positive colonies were preliminary identified
by yielding a PCR product of the expected
size.
To verify the correctness of the
recombinant plasmid and to prevent false
positive caused by PCR amplification, double
digestion of the preliminary identified
recombinant plasmid pET28a-pncA was
carried out.
Immunoblot assays were performed using the
Immuno-Blot Assay from Bio-Rad, using
colorimetric detection with alkaline phosphatase.
The proteins were electrophoretically transferred
from the polyacrylamide gel to PVDF membrane
using a semidry blotting apparatus. The primary
antibody was a mouse anti-His antibody provided
by Upstate Biotechnology. The secondary
antibody was a rabbit anti-mouse IgG(H+L)
antibody conjugated to alkaline phosphatase .
III. Results
A. Identification of Recombinant
Plasmid pET28a-pncA.
The genomic DNA of M.tuberculosis
strain H37Rv was obtained from bacterial
cultures. PCR was amplified with pncA-5’
and pncA-3’ primers and high fidelity pfu Tag
DNA polymerase to obtain 561 bp pncA gene.
The amplified product was analyzed by 1%
(w/v) agarose gel electrophoresis (Figure.1).
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Shi et al: Prokaryotic expression, purification of Mycobacterium tuberculosis PZA-resistant protein
The digestion products showed the vector
fragment of about 5000bp and the insertion
gene pncA fragment of about 600 bp as
expected. Positive clone was chosen to be
sequenced (Figure.3). The DNA sequence
was consistent with the reported GenBank
pncA gene sequence (GenBank accession
accession number U59967).
Figure 6. Western blot detection of purified fusion
protein.
B. Pyrazinamidase Recombinant Protein
Expression,
Purification
and
Immunoblotting Analysis.
The recombinant expression plasmid was
transformed into E.coli BL21 cells for
prokaryotic expression. The vector alone was
also introduced into E. coli as a control. To
optimize the PZase expression condition and
to prevent inclusion body protein formation,
recombinant E.coli was each grown in four
batches by shaking conditions for
approximately 4-6h. Recombination protein
could be highly expressed in supernatant
solution without inclusion body protein
formation at 30℃ or at 37℃. Moreover, the
amount of target protein increased with
extension of the induced period. The PZase
fusion protein migrated on 12% SDS-PAGE
gel as an about 20kDa protein (Figure.4).
The size of the fusion protein was consistent
with the predicted molecular mass of the
PZase fusion protein.
Figure 2: Identification of the recombinant plasmid by
colony PCR. M:50bp Ladder Marker; 1-3:PCR
production of positive colony; 4-5:PCR production of
negative colony
Figure 3 ： Identification of the recombinant plasmid by
restriction enzyme cleavage.
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Gene Therapy and Molecular Biology Vol 15, page 190
Figure 4: SDS-PAGE identification of pET28a-pncA
expressed in E.coil BL21. M: Protein Marker; 1:E.coil
BL21 (transformed with pET28a-pncA ) protein
without induction; 2-3: E.coil BL21 (transformed with
pET28a-pncA ) protein induced at 37 ℃ by IPTG for 4
and 6h; 4-5: E.coil BL21 (transformed with pET28apncA) protein
Figure 5: SDS-PAGE identification of purified fusion
protein.
To
determine
whether
the
predominantly expressed protein is the
PZase，the lysate supernatant with 6×His tag
was subjected to Ni2+-NTA agarose column
for affinity chromatography purification, and
the column was washed with 200 mmol/L
imidazole gradient elution buffer. The
purified protein was subjected to SDS-PAGE
followed by Coomassie blue staining and
western blot. Coomassie staining indicated
visible band the position 20kDa around,
which is consistent with the predicted size of
the PZase (Figure.5).
To identify whether the purified
protein was His-tag fusion protein, the
purified product was detected by western blot
with anti-His monoclonal antibody. As
expected, the target protein (20 kD) was
detected
by
western-blot
(Figure.6).
Altogether, these results demonstrated that
the purpose protein was the His-tagged PZase
protein.
IV. Discussion
The antituberculous activity of
pyrazinamide （ PZA ） was discovered in
1952, but it played an important role on
accelerating the effect of therapy when used
in combination with isoniazid and
rifampin(Chang, Leung et al. 2012; Fonseca,
Marsico et al. 2012; Grosset, Almeida et al.
2012).These observations allowed for the
shortening of the therapy for tuberculosis
from 9 to 6 months and made PZA the third
most important drug in the modern therapy
of tuberculosis. The ability of pyrazinamide
to shorten the therapy seems to correlate
with its ability to kill a special bacterial
population with low metabolic activity
residing in acidic pH environment that are
not killed by other TB drugs and also its
ability to kill old non-growing bacilli more
effectively than young bacilli(Mitchison
1985; Zhang, Permar et al. 2002).
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Shi et al: Prokaryotic expression, purification of Mycobacterium tuberculosis PZA-resistant protein
Pyrazinamide (PZA) is an effective
first-line drug for the treatment of
tuberculosis (TB). It is a pro-drug that
requires conversion into its active form,
pyrazinoic acid, by the bacterial enzyme
pyrazinamidase (PZase), which is encoded
by the 561-nucleotide pncA gene(Scorpio
and Zhang 1996; Zhang and Mitchison
2003). Mutations in pncA result in lost or
reduced PZase activity, and such mutations
are thus considered to be the primary
mechanism
of
PZA resistance
in
Mycobacterium tuberculosis(Scorpio and
Zhang 1996; Hirano, Takahashi et al. 1997).
pncA gene mutations have been detected in
PZA-resistant clinical isolates of M.
tuberculosis, as well as in Mycobacterium
bovis, which is naturally resistant to the
drug(Scorpio and Zhang 1996).
The susceptibility testing of M.
tuberculosis to PZA is technically difficult
because PZA is only active at acidic
pH(Hirano, Takahashi et al. 1997; Cheng,
Thibert et al. 2000). Unfortunately, a low pH
level (pH 5.5) that is required prevents some
of the M. tuberculosis clinical isolates from
growing in Lowenstein-Jensen Medium and
in Middlebrook 7H10 agar medium. Other
approaches have been attempted to detect
the presence or absence of the
pyrazinamidase (PZase) function. PZA
resistant M. tuberculosis strains lose PZase
activity. There is a positive correlation
between the loss of PZase activity and the
development of PZA resistance in M.
tuberculosis strains(McClatchy, Tsang et al.
1981; Trivedi and Desai 1987). It takes more
than 2 weeks to obtain results by the
conventional PZase assay method(Wayne
1974).
A number of rapid and simple methods
based on the PCR technique have been
developed for the detection of a mutation on
the gene responsible for the drug resistance.
PCR
single
strand
conformational
polymorphism and molecular beacon
sequence analysis are suitable for the
detection of a limited number of mutations
on specific genes. PCR product direct
sequencing and DNA microarray can detect
many mutations at the same time. The latter
two were suitable for the detection of PZAresistant M. tuberculosis because the
mutations responsible for PZA resistance
were dispersed on the pncA gene. However,
as these methods require costly apparatus,
such as a DNA sequencer or laser scanner,
they can not be done in the small clinical
laboratory.
To date reformed E. coli has been used
extensively as the cellular host for foreign
protein expression due to its rapid growth
rate, capacity for continuous fermentation,
and relatively low cost. The pET28a used in
our study is a powerful system yet
developed for the cloning and expression of
recombinant proteins in E.coil. In this study,
the pncA gene was cloned into prokaryotic
expression plasmid pET28a containing six
histidine
residues
by
recombinant
technology.
The
target
protein,
pyrazinamidase, was highly expressed in
host stain, and the molecular weight of the
recombinant protein is about 20kDa, which
is consistent with the predicted size of the
PZase. His-fusion proteins are purified by
Ni2+-NTA
agarose
column
affinity
chromatography. This study lays an
experiment foundation for the understanding
of the molecular basis of pyrazinamide drugresistance in M.tuberculosis.
In this study we expressed and purified
the PZase using affinity chromatography,
and the activity of a recombinant PZase
should not be different than a native protein
in M. tuberculosis because this is a strict
cytosolic enzyme, thus its folding in E. coli
is expected to be similar in M. tuberculosis.
In future, pncA can be amplified by PCR
from the sputum specimens with positive
smear for acid-fast bacilli, and then the
PZase can be expressed in vitro from cloned,
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Gene Therapy and Molecular Biology Vol 15, page 192
recombinant DNA. Then these expression
products will be used directly for PZase
activity assay based on the color developed
due to the reaction of ferrous ammonium
sulfate with pyrazinoic acid, which was
converted from PZA by the in-vitro
synthesized PZase. A sample will be
considered as resistant if its synthesized
PZase shows any reduction in activity/color
absorption compare with that of the PZase
from M. tuberculosis H37Rv, otherwise, as
susceptible.
Therefore, this study also lays the
substantive foundation for the development
of a simple method which enable rapid PZA
susceptibility testing based on PCR
amplificationof pncA gene and an in-vitro
system. Therefore，this study may have the
potential of being a direct test for sputum
specimens bypassing the lengthy cultivation
of tubercle bacilli.
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