Ahmad 2019 J. Phys.: Conf. Ser. ks1341 032011

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The 3rd International Conference On Science IOP Publishing
Journal of Physics: Conference Series 1341 (2019) 032011 doi:10.1088/1742-6596/1341/3/032011
Cloning and expression of recombinant protein CFP21 from

Mycobacterium tuberculosis as a tuberculosis vaccine candidate
A Ahmad1,2*, R Agus3, M N Massi4, I Handayani5 and H Karim6

1
Chemistry Department, Mathematics and Natural Science Faculty, Hasanuddin Univesity,
Perintis Kemerdekaan Street Km. 10 Tamalanrea, Makassar 90245, Indonesia
2
Laboratory of Centrer of Research and Science Development, Faculty of Natural Sciences,
Hasanuddin University, Perintis Kemerdekaan Street Km. 10, Makassar 90245, Indonesia
3
Biology Department, Mathematics and Natural Science Faculty, Hasanuddin Univesity, Perintis
Kemerdekaan Street Km. 10 Tamalanrea, Makassar 90245, Indonesia
4
Microbiology Department, Faculty of Medicine, Hasanuddin Univesity, Perintis Kemerdekaan
Street Km. 10 Tamalanrea, Makassar 90245, Indonesia
5
Clinical Pathology Laboratory, RSWS Hospital, Perintis Kemerdekaan Street Km. 10
Tamalanrea, Makassar 90245, Indonesia
6
Department of Pharmacy, School of Pharmacy YAMASI, Jl. Mapala 2 Blok D5 No.10
Makassar 90222, Indonesia
*
Email : [email protected]
Abstract. Increased resistance to TB drugs, may render vaccine development a more effective
approach to stop or reduce TB epidemics. The antigen Culture Filtrat Protein Filtrat 21
(CPF21) is an immudominant protein encoded in RD 2 region of the Mycobacterium
tuberculosis genome, capable of obtaining a strong hypersensitivity reaction and to induce very
high interferon-gamma (IFN-γ) responses in patients with tuberculosis. In order to construct the
recombinant plasmid pGEM-T Easy-CFP21 and express it in E. coli BL21, the CFP21 gene was
amplified from M. tuberculosis H37Rv genomic DNA using PCR in vitro, and inserted into the
pGEM-T Easy cloning vector. The recombinant plasmid was then transformed into E. coli
JM109, followed by plasmid extraction, PCR amplification, and DNA sequencing. The correct
recombinant CFP21 gene was subcloned into expression vector pGEX-2TK and transformed
into E. coli BL21 strain. The white recombinant colony was selected, cultured, induced with 50
µM IPTG, and identified using SDS-PAGE electrophoresis method. These results demonstrated
that CFP21 gene has been constructed and expressed successfully. The molecular weight was
about 47 kDa as the fusion protein GST-CFP21 and expressed as insoluble protein. In
conclusion, the target gene CFP21 has been cloned into host E. coli BL-21 strain and expressed
successfully. In the future, the purified recombinant fusion protein GST-CFP21 paves the way
for TB diagnosis and vaccine development.
1. Introduction
Tuberculosis (TB), malaria and AIDS are the of the three major infectious diseases globally, with 9
million new cases and 2 million deaths per year. In Indonesia, 70% of TB cases occur in the
productive age that causes enormous social and economic burden. Mycobacterium tuberculosis has
infected about one third of the population worldwide. Every year, around 8 million infected people
Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution
of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
Published under licence by IOP Publishing Ltd 1
develop active disease and 2 million of them die from tuberculosis. Around 10% of TB cases in the
world are found in Indonesia and this puts Indonesia at third highest TB burden country after India
and China[1].
Management of TB is increasingly difficult with the emergence of drug resistant strains. Multi-
drug resistant M. tuberculosis (MDR-TB) strain is known to be resistant to two main antibiotics that
are often used, namely isoniazid and rifampicin. Furthermore, M. tuberculosis strains that show
resistance to additional TB drugs (Extensive drug-resistant M. tuberculosis, XDR-TB) were found.
Besides isoniazid and rifampicin, XDR-TB strains are also resistant to fluoroquinolones and
injection drugs e.g. kanamycin, amikacin, or capreomycin [2]. The emergence of these strains has
caused new issues in TB management globally and made WHO proclaim TB as a worldwide crisis.
Due to increased pathogen resistance to TB drugs, the development of more effective vaccines is
believed to be a necessary solution to stop or reduce the TB epidemic. Some criteria for an effective
TB vaccine are the capabilities to provide better immunity than BCG; to stimulate a quick response
for protection against TB; to reduce pain in people affected by TB; and to prevent TB reactivation
[2,3].
The formation of antibodies to the M. tuberculosis antigen takes a long time because the
tuberculosis bacterial infection is a slow type hypersensitivity reaction and involves the cellular
immune responses more than the humoral immune responses in its pathogenesis. Thus, antibody-
based examination cannot detect TB disease at the initial stage of infection [4]. At present, several
types of antigens from M. tuberculosis are being examined as TB vaccine candidates. ESAT-6 is an
antigen that is widely used in several previous studies as TB vaccine candidates. In its development,
the ESAT-6 antigen is an important protein used in the diagnosis to distinguish between non-TB and
TB patients (who have been vaccinated with BCG). Thus, ESAT-6 is no longer used in the
development of TB vaccines [5,6,7]. In our previous study, we successfully cloned and produced
MPT83 recombinant antigen using the E. coli BL-21 cell as a host [8]. Besides the MPT83 antigen,
another antigen, CFP21, is found in the M. tuberculosis genome. CFP21 antigen is an
immunodominant protein encoded in the RD2 region in the M. tuberculosis genome and proven
capable of inducing IFN-γ released from cytotoxic T lymphocytes of TB patients, so that the antigen
is very potential as a vaccine candidate [9,10].
Based on the description above, a study of cloning and expression of CFP21 antigen with
recombinant DNA technology has been carried out as a candidate for an effective TB vaccine in the
future. Thus the antigen is expected to decrease the cost of making TB vaccines, provide immunity
against active TB patients in productive age, which in turn can reduce the morbidity and mortality
caused by TB.
2. Materials and methods

2.1 Materials
The materials used in this study were E. coli strain JM109 and BL21, plasmid pGEM-T Easy and
pGEX2TK, LB medium (Luria Bertani), ampicillin, IPTG, NaCl, KCl, Na 2HPO4, KH2PO4, Loading
buffer, Acrylamide / bis solution (Bio-rad), Tris Base (Calbiochem), SDS (Sigma), Ammonium
Persulfate (Sigma), Tetramethylethylenediamine (Sigma), Glisine (Sigma), protein marker (Thermo
Scientific), Methanol (Merck), Glacial Acetid Acid, Geneaid Kit, Qiagen Kit, master mix go Tag
green Kit, loading dye, DNA marker 100 bp, Bacto trypton, Bacto agar, Bacto yeast, comma brilliant
blue (Bio-rad), Filter tip (Genfollower), and Barrier tips (Multiguard).
2.2 Instrumentations
The instruments used in this study were thermo cycle PCR machines, shakers incubator (Heidolph
Duomax 1030), centrifuges (Profuge), sonicators, Mini Protean II Bio Rad, Electrophoresis (Bio-rad),
autoclaves (Hirayama), laminar air flow (Esco Ductless Fume Cabinet), Erlenmeyer (Pyrex), scale
(Kern 440-47N), micropipette (Bio rad), beaker (Pyrex), eppendorf tube (Axygen), Hot plate
(Memmert), and waterbath (Memmert).
2
2.3 Methods
2.3.1 Isolation of M. tuberculosis DNA genome. Isolation of M. tuberculosis H37Rv strain genomic
DNA was carried out by the modified Boom method. Into 100 µl of sample, 900 µL of L6 lysis buffer
was added, and centrifuged at 12,000 rpm for 10 minutes. Then the supernatant was added with 20 µl
celite, followed by centrifugation at 10,000 rpm for 15 seconds. The precipitate was washed with 1
mL of L2 buffer twice. Then washed with 1 mL of 70% ethanol, and 1 mL of acetone twice. The
genomic DNA deposition is dissolved in buffer TE pH 7.4 [11].
2.3.2 Amplification of CFP21 gene coding 21 kDa antigen. Amplification of the CFP21 gene was
carried out with the PCR technique using a pair of specific primers. The primary sequence used in this
study was CFP21-F: 5'-GCCGGGATCCATGCATTTCG-3 'and CFP21-R: 5'-GGCAAGCTT
CTAAGTGTCTG-3' [9,10] BamHI restriction enzyme sites (bold underline) are added before the
ATG start codon in the primary forward and HindIII (bold underline) sites are added after the codon
stops at the reverse primer. Components of the PCR reaction of 25μL were made by reacting 1 µL of
the forward primer (10pmol), 1 µL of reverse primer (10pmol), and 3µL of isolated genomic DNA and
fulfilled with Master Mix go Taq green. The PCR conditions used were pre-denaturation of 94oC for 5
minutes, initial denaturation at 94oC for 30 seconds, primer attachment (annealing temperature) at
55oC for 30 seconds, and elongation at 72 oC for 30 seconds. Lengthening the final DNA fragment at
72oC for 7 minutes and the PRC reaction was carried out in 30 cycles.
2.3.3 Cloning CFP21 gene to cloning vctor of pGEM-T Easy. The amplified CFP21 gene was cloned
to the pGEM-T Easy cloning vector. PCR products were isolated from agarose gel with GFX column
KIT and then cut with BamHI / HindIII restriction enzyme, ligated into the pGEM-T Easy cloning
vector after being cut with the same restriction enzyme. The ligation results were transformed into E.
coli JM109 and selected by selection media containing ampicillin, X-Gal and IPTG. Colonies
containing pGEM-T Easy with inserted CFP21 DNA should be white in color and confirmed with
colony PCR technique by Qiagen plasmid kit [12]. Determination of the nucleotide sequence of the
CFP21 gene as a DNA insertion was performed using a DNA sequencer (Applied Biosystems ABI
310 Model). The DNA sequencing results were then homologized with the order in the data bank to
determine the homology level with the help of the BLAST program on the NCBI website
(http://www.ncbi.nlm.nih.gov) and Bioedit application v.7.0.9 (https:// bioedit.software.
informer.com/7.0.9/).
2.3.4 Expression of recombinant protein CFP21 in E. coli strain BL21. The CFP21 gene fragments
that had been confirmed by the DNA sequencing technique were subcloned into the pGEX2TK
expression vector to produce plasmid pGEX2TK-CFP21. E. coli BL21 (DE3) cells were transformed
with the recombinant plasmid pGEX2TK-CFP21 containing the CFP21 gene to produce fusion protein
GST-CFP21. Recombinant E. coli BL21 clones that had carried plasmid constructs pGEX2TK-CFP21
were inoculated into 15 mL of LB media containing 100 μg/mL ampicillin and then incubated for 10
hours in a shaker incubator at 37 oC at 250 rpm. Then 2% (300 μl) of E. coli BL21 culture was
transferred into 10 mL of new LB media which was added with 100 μg/mL ampicillin, incubated for 2
hours in a shaker incubator at 37 oC. A total of 5 mL of bacterial culture was transferred to a test tube
that did not contain IPTG (CFP21 non-induction, - IPTG). The remaining bacterial culture (5 mL)
underwent protein expression by 50 µM IPTG induction for 6 hours (CFP21 by induction, + IPTG).
E.coli BL21 culture in each test tube was centrifuged at a 14,000 rpm for 5 minutes, to separate the
supernatant from the cell pellet. Proteins were extracted from cell pellets which were resuspended in
PBS 1X buffer pH 7.4 containing (0.8 gr NaCl, 0.02 gr KCl, 0.144 gr Na 2HPO4, 0.024 gr KH2PO4 and
100 mL ddH2O) and sonicated. Bacterial walls were ruptured with a 30-second recurring sonication
technique 3 times with a power of 20 kHz. The fractured bacterial cell suspension was centrifuged at a
speed of 14,000 rpm at 4oC for 1 minute to separate recombinant proteins from bacterial cell debris.
Analysis of recombinant protein expression in the supernatant and cell pellet used sodium dodecyl
3
sulphate-polyacrilamide gel (SDS-PAGE) electrophoresis technique. The results of CFP 21

recombinant protein were analyzed by comparing the results of protein bands on polyacrylamide gels
with protein markers. Then the data was presented in the form of images and descriptively narrated.
3. Results and discussion
3.1 Isolation of M. tuberculosis DNA genome
The CFP21 antigen was isolated from M. tuberculosis genomic DNA H37Rv using the modified
Boom method with a heating-centrifugation/boiling technique using DNA extraction kit with
consideration of purity and primary specific attachment specificity at the amplification stage of the
target gene region. The number of samples used were 4 colonies from M. tuberculosis, strain H37Rv.
Of the four samples, each DNA concentration was measured using a nanodrop spectrophotometer at
absorbance of 260 nm (data not shown). Data on agarose electrophoresis analysis of M. tuberculosis
strain H37Rv genomic DNA and negative control samples are shown in Figure 1.
Figure 1. Electrophoresis results of genomic DNA isolated from M.

tuberculosis strain H37Rv (line 1-4) and negative control (line 5).
3.2 Amplification of CFP21 gene encoding 21 kDa antigent

The results of amplification of CFP21 gene with PCR using specific primer are shown in Figure 2.
1 2 3 M
Figure 2. The results of PCR amplification (column 2), purification

of CFP21 gene product DNA using Purification Kit (Qiagen) (column
3), negative control (column 1), and DNA marker (column M)
4
From Figure 2, it is shown that the PCR product is in the same migration position with the 600 pbs
DNA marker, precisely measuring 608 bps. This is in accordance with the data from GenBank (No.
access: NC_000962.3) that the CFP21 gene encodes 21 kDa antigen consisting of 608 bps. The PCR
product then becomes the target gene to be ligated into the pGEM-T Easy cloning vector.
3.3 Construction of CFP21 gene into cloning vector pGEM-T Easy

Cloning was performed in order to obtain CFP21 genes in large quantities. In this study we used the
pGEM-T Easy cloning vector because it has the Origin of Replication (ORI) for insertion DNA
fragments. In addition, the pGEM-T Easy vector has an ampicillin (Ampr) gene and lacZ gene site that
plays a role in blue-white screening during transformation into competent cells of E. coli JM 109.
DNA ligation result from CFP21 gene encoding 21 kDa antigens insertion into pGEM-T Easy cloning
vectors to obtain recombinant plasmids (Figure 3).
Figure 3. The result of electrophoresis of agarose gel in ligation

process of CFP21 gene into pGEM-T Easy cloning vectors.
Column 1: pGEM-T Easy vector; Column 2: recombinant plasmid
pGEM-T Easy-CFP21, each had been restricted by
BamHI/HindIII restriction enzyme. Column 3: DNA marker
From the Figure 3 above, it appears that the CFP21 gene insertion has been successfully ligated to
the pGEM-T Easy cloning vector. This is evident in column 2 where pGEM-T Easy-CFP21 which has
been restricted with BamHI and HindIII produced two bands of DNA fragments of 3015 pbs and 608
bps, so it can be concluded that the plasmid pGEM-T Easy has been easily inserted with the CFP21
gene.
3.4 Transformation of pGEM-T Easy-CFP21 into competent cell (E. coli JM109)
The transformation was carried out using the competent cell E. coli JM109 which functioned as a cell
host that would multiply the recombinant plasmid pGEM-T Easy-CFP21. The transformation resulted
in white bacterial colonies and blue colonies on agar LB plate medium containing X-gal, IPTG and
ampicillin (Figure 4). White colonies indicate that the insertion DNA has been successfully inserted
5
into the vector, while the blue colony means that the insertion DNA has not been successfully inserted
to vector (self-ligation). Agar LB plate medium that has been added with ampicillin antibiotics,
isopropyl β-D-1-thiogalactopyranoside (IPTG), and X-Gal, can be used as a selection medium for
transformed competent cell growth. Ampicillin serves as the first selection medium, because the
bacteria that will grow are bacteria that have ampicillin resistance genes, namely E. coli containing
vector pGEM-T Easy. The addition of IPTG was intended as a transcription inducer in the lac operon
gene lacZ.
Figure 4. E coli cells growth where blue colony is self-ligated

and white colony contains pGEM-T Easy-CFP21 recombinant
plasmid.
The lacZ gene will encode an β-galactosidase enzyme which functions to break down lactose into
glucose and galactose. The presence of the β-galactosidase enzyme in this method is detected by the
breakdown of the colorless X-gal substrate (5-bromo-4chloro-3-indolyl-β-D-galactactoside) to blue
galactose and 5-bromo-4-chloroindigo [13]. The CFP21 insertion into pGEM-T Easy cloning vector
will disrupt the expression of β-galactosidase enzyme, and therefore, bacterial colonies successfully
transformed with the pGEM-T Easy-CFP21 plasmid will appear white. Cells not transformed with this
particular plasmid will remain blue.
3.5 Isolation and characterization of pGEM-T Easy-CFP21 recombinant plasmid

Analysis of CFP21 gene insertion DNA was carried out by colony PCR technique. The PCR product
shows that there is one DNA band with a size of 608 bps. The PCR results prove that plasmid DNA
from the white colonies contained insertion DNA in the form of the CFP21 gene (Figure 5A columns
4-6), whereas the blue colony PCR results this band did not appear (Figure 5A columns 1-3).
Recombinant plasmid isolation from three white colony in Figure 5A, was carried out based on the
QIAprep Spin Miniprep Kit from Qiagen (USA). The results obtained can be explained by
electrophoresis technique and visualized on agarose gel (Figure 5B column 1-3).
6
Figure 5. A. Colony PCR CFP21 gene products as insert DNA, column1-3 were derived
from blue colonies and column 4-6 were derived from white colonies. White colonies
were picked up and cultured in LB liquid media followed by CFP21 gene amplification
by PCR and then analyzed on agarose gel electrophoresis. B. Visualization of
recombinant plasmid isolation from three white colony in A panel.
3.6 Sequencing of pGEM-T Easy- CFP21 Recombinant Plasmid

The results of sequencing of the genetic code of cDNA fragments from CFP21 cloned into the
pGEM-T Easy plasmid resulted in recombinant plasmid pGEM-T Easy-CFP21 using the Bigdye
Terminator method with the Applied ABI PRISM 310 Biosystem sequencing tool, which was then
processed with Bioedit application. v.7.0.9. The sequencing result is shown Figure 6. The size of the
cDNA in full sequencing of the gene encoding the CFP21 protein is 654 bps (Figure 6), with 217
amino acid residues starting from the amino acid methionine (M) to stop codon.
7
Figure 6. Nucleotide sequence and amino acid prediction from CFP21 gene
3.7 Expression of CFP21 gene to E. coli BL21 cell

This study started by growing E. Coli BL21 cells carrying recombinant plasmid pGEX2TK-CFP21. E.
Coli BL21 cells were taken from the cloning of the previous CFP21 recombinant protein which was
characterized by the growth of white colonies on LB (Luria Bertani) media. Blue colonies show no
DNA insertion in the vector plasmid. White colonies are cells that do not have the activity of the
8
enzyme β-galactosidase. This is due to the insertion of DNA fragments located between the lac Z gene
so that DNA fragments will activate the expression of the lac Z gene [14]. In order to confirm that
bacteria grown on ampicillin media were successfully inserted into the CFP21 gene, the
transformation process was carried out. The white colonies formed indicate that the bacterial clones
were successfully inserted in the CFP 21 gene. The white colonies were then used as candidates for the
production of CFP21 recombinant protein as future TB vaccine candidates.
The growth of E. coli BL21 pGEX2TK-CFP21 recombinant plasmid carrier was characterized by
the occurrence of discoloration in the media to become cloudy. According to [15] E. coli is a
prokaryote organism that is most widely used as a host cell in research. E. coli bacteria have the ideal
characteristics as host cells which can reproduce themselves in a short and stable time, having high
expression levels, non-pathogenic and most importantly can be introduced with various foreign genes
that encode target proteins.
Figure 7. Growth of E. coli strain BL21 containing recombinant plasmid pGEX2TK-

CFP21 in (A) Agar LB plate medium and (B) liquid LB culture medium. (1) Growth and
expression of GST-CFP21 recombinant fusion induced by IPTG. (2) Expression of
recombinant proteins not induced by IPTG. (3) Control medium without transformed by
plasmid.
Growth of E. coli BL21 that contain recombinant plasmid pGEX2TK-CFP21 in liquid LB media
containing ampicillin caused turbidity in culture media, as shown in Figure 7B line 1 and 2. Cell lysis
was carried out by ultrasonication method. The physical cleaning method using ultrasonic waves is
commonly known as the sonication method. The Sonication method is a cell-breaking method that
utilizes vibrations from sound waves with high frequency to lyse cells. Proteins are extracted from
deposits of cells resuspended in the lysis buffer and are sonicated until dissolved. Breaking bacterial
walls with a one minute recurring sonication technique. Sonication is carried out in lysis buffer
solution at 20 kHz until the liquid looks clear. Then centrifugation was performed to separate
recombinant proteins and bacterial cell walls. Cell deposition as an insoluble fraction and supernatant
containing insoluble protein was analyzed using SDS-PAGE method.
The SDS-PAGE method is a technique for separating polypeptide chains in proteins based on
charged molecular weight under the influence of an electric field [12]. Before SDS-PAGE is carried
out, proteins are first converted from globular structures into linear structures with denaturation. This
analysis with SDS-PAGE uses polyacrylamide gel consisting of stacking gel and separating gel.
Stacking gel has a well that serves as a place to place the sample while separating gel is where the
protein will move towards the anode.
The results of the electrophoresis were then immersed in a staining solution (commassie brilliant
blue R-250). Commassie brilliant blue R-250 functions as a protein blue giver in electrophoresis gel.
The positively charged Commassie brilliant blue R-250 will bind to negatively charged proteins
through electrostatic forces.
Recombinant protein GST-CFP21 expression with IPTG induction for 6 hours clearly shows the
9
blue fusion protein band on SDS-PAGE with a molecular weight of 47 kDa derived from a
combination of GST (26 kDa) and CFP21 (21 kDa), whereas expression without IPTG induction
does not show this band (Figure 8). This proves that the inducer (IPTG) greatly determines the success
of recombinant protein expression. IPTG (isopropyl-β-D-thiogalactopyranoside) is a common inducer
used for the induction of expression of recombinant proteins as well as induction materials that can
increase the expression of target proteins in E. coli cells.
The formation of GST-CFP21 protein when induced with IPTG proved the reaction between
protein samples and loading buffer. This is influenced by the content of glycerol and bromophenol
blue in the loading buffer. Glycerol serves to stabilize the weight of the sample to settle at the bottom
of the sample well, while blue bromophenol serves as tracking dye to mark the farthest limit of the
movement of protein samples at electrophoresis so that the protein band appears blue. This is
consistent with [15] statement that IPTG compound cannot be metabolized by cells, so the induction
rate is always constant even though there is an addition of IPTG. The results of the analysis by SDS-
PAGE electrophoresis method in samples induced with 50 µM IPTG showed that GST-CFP21
recombinant protein was expressed in the insoluble (pellet cell) fraction. The formation of this
insoluble protein may occur because the GST-CFP21 fusion protein has a molecular weight of 47 kDa.
The study of Hasegawa et al (2002) [16] comparing the molecular weight of several recombinant
proteins with their level of solubility, shows that proteins with a size below 30 kDa require fusion
with solubelitas and protein enhancers tend to be expressed in soluble. Whereas the insoluble form is
due to above 30 kDa protein being expressed excessively and heterologically in E. coli, so that it still
allows the inclusion of the body (inclusion body protein) which is an insoluble protein which blends
with E. coli cell debris. The next research will be sought methods of expression of fusion protein GST-
CFP21 to become a soluble protein which can be separated from E.coli cell debris.
Figure 8. SDS-PAGE (10%) analysis results (1) Protein Marker (M), which is
arranged from top to bottom: 212kDa; 116 kDa; 97.4 kDa; 66.2 kDa; 45 kDa; 31
kDa; (2) Expression of GST-CFP21 recombinant fusion protein induced by 50 µM
IPTG for 6 hours (+ IPTG); (3) Expression of GST-CFP21 recombinant fusion
protein without induced by IPTG (- IPTG).
10
4. Conclusion
From the results and discussion described above it can be concluded that: (1) CFP21 antigen from the
bacterium M. tuberculosis strain H37Rv was successfully amplified by PCR technique, which
produced a partial band with the size of 608 bp star ATG codon. (2) At the cloning stage of CFP21
gene into pGEM-T Easy vector, it produces pGEM-T Easy-CFP21 recombinant plasmid with a formed
band length of 3623 bps. (3) The CFP21 gene successfully sub-cloned into the expression vector
pGEX-2TK produces plasmid pGEX-2TK-CFP21 and is expressed in E coli BL-21 cells to produce
fusion proteins with a molecular size of 47 kDa as a fusion protein GST-CFP21.
Acknowledgements
The authors thanks for Mrs. Rufika Shari Abidin for editorial reading of the manuscript. The authors
thanks also to Mrs. Rina for invaluable help in technical assistance and sample DNA and recombinant
vector preparation in the Nechri Laboratory, RSWS hospital, Hasanuddin University, Indonesia. This
work was supported in part by a Grant-in-Aid by National Applied Research Project 2019 by contract
number 1740/UN4.21/PL.01.10/2019 of Directorate of Career and Competency of Human Resourses
of the Ministry of Research, Technology, and Higher Education of the Republic of Indonesia.
References
[1] WHO 2016 BCG – The Current Vaccine for Tuberculosis Initiative for Vaccine
Research World Helath Organization http://www.who.int (accessed on 14 May 2018)
[2] WHO 2018 Global Tuberculosis Report 2017 www.whoint/tb/publications/globalreport/
(accessed on 19 May 2018)
[3] Paridah and Kaufman 2010 The Quest for Biomarkers in Tuberculosis Drug Discov Today
15: 148–157 doi: 10.1016/j.drudis.2009.10.005
[4] Mathur M L Lobue P A and Catanzaro A 1999 Evaluation of a Serologic Test for the Diagnosis
of Tuberculosis. The International Journal of Tuberculosis and Lung Disease 732-5
[5] Dietrich R Moravek M Bürk C Granum P E Märtlbauer E 2005 Production and Characterization
of Antibodies against Each of The Three Sub Units of the Bacillus cereus Non Hemolytic
Enterotoxin Complex Appl Environ Microbiol 71: 8214–8220
[6] Agus R 2009 Potensi Antigen ESAT-6 Rekombinan dari Mycobacterium tuberculosis Sebagai
Kandidat Vaksin Tuberkulosis. Digital Library. Indonesian Science & Technology Available
at: http://elib.pdii.lipi.go.id
[7] Skjot RLV Oettiger T Roswnkrands I Ravn P Brock I Jacobson S et al 2000
Comparative Evaluation of Low–Molecular–Mass Protein from Mycobacterium tuberculosis
Identifies Members of The ESAT – 6 Family as Immune Dominant T – cells antigen Infect
Immun 68:214-20.
[8] Ahmad A Agus R Massi M N Natzir R Madhyastha R Madhyastha H K and Maruyama M
2018 Cloning and Expression of MPT83 Gene from Mycobacterium tuberculosis Local
Strain from Clinical Isolates In E. coli BL21 As Vaccine Candidate of Tuberculosis: A
Preliminary Study J. of Genetic Eng. and Biotech 16(2) 335-340
[9] Fu R Chun W Chunwei S Mengji L Zhengming F Jia L Fang W and Xiongling F 2009 An
Improved Whole Blood Gamma Interferon Assays Based On CFP21- MPT64 Fussion
Protein Clinical and Vaccine Immunology 16(5): 686-690
[10] Fayaz A G Mir F A Verma I Sharma P Khuller G 2006 Expression and Purification of The
Mycobacterium tuberculosis Complex-Restricted Antigen CFP21 to Study its
Immunoprophylactic Potential in Mouse mode Protein Expression and Purification 48(2):
274-280
[11] Hatta M and Smits H L 2007 Detection of Salmonella typhi by Nested Polymerase Chain
Reaction in Blood Urine and Stool Samples American J. Tropical Medicine Hygiene 76:139-
143
11
[12] Sambrook J Fritsch E F & Maniatis T 1989 Molecular Cloning: A Laboratory Manual 2nd Ed.
(New York: Cold Spring Harbor Laboratory Press)
[13] Madigan M T and Martinko J M 2005 Brock Biology of Microorganisms 11th ed. (New Jersey:
Prentice Hall)
[14] Wang C Chen Z Fu R Zhang Y Chen L Huang L Li Shi C and Fan X 2011 A DNA
Vaccine Expressing CFP21 and MPT64 Fusion Protein Enhances BCG-Induced
Protective Immunity against Mycobacterium Tuberculosis Infection in Mice Med
Microbiol Immunol 200(3):165-75
[15] Donovan R S Robinson C W & Glick B R 1996 Review: Optimizing Inducer and Culture
Conditions for Expression of Foreign Proteins Under The Control of The Lac Promoter
Journal of Industrial Microbiology 16: 145-154
[16] Hasegawa N Takao M Koudou I Kazuhiro Y Thomas H L Samuel M Janis D M Salman H S
2002 New Simple and Rapid Test for Culture Confirmation of Mycobacterium
Complex: a Multicenter Study Journal of Clinical Microbiology 40(3): 908–912
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Ahmad 2019 J. Phys.: Conf. Ser. ks1341 032011

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Journal of Physics: Conference

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Cloning and expression of recombinant protein CFP21 from

A Ahmad1,2*, R Agus3, M N Massi4, I Handayani5 and H Karim6

2. Materials and methods

sulphate-polyacrilamide gel (SDS-PAGE) electrophoresis technique. The results of CFP 21

Figure 1. Electrophoresis results of genomic DNA isolated from M.

3.2 Amplification of CFP21 gene encoding 21 kDa antigent

Figure 2. The results of PCR amplification (column 2), purification

3.3 Construction of CFP21 gene into cloning vector pGEM-T Easy

Figure 3. The result of electrophoresis of agarose gel in ligation

Figure 4. E coli cells growth where blue colony is self-ligated

3.5 Isolation and characterization of pGEM-T Easy-CFP21 recombinant plasmid

3.6 Sequencing of pGEM-T Easy- CFP21 Recombinant Plasmid

3.7 Expression of CFP21 gene to E. coli BL21 cell

Figure 7. Growth of E. coli strain BL21 containing recombinant plasmid pGEX2TK-

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