Vol.1, No.1, 1-7 (2011) Open Journal of Immunology
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJI/
Successful expression and purification of dppd, using a
codon optimized synthetic gene*
Suely S. Kashino, Antonio Campos-Neto#
The Forsyth Institute, Global Infectious Disease Research Center, Boston, USA; #Corresponding Author: acampos@forsyth.org
Received 14 May 2011, revised 8 June 2011, accepted 17 June 2011.
DPPD (Rv0061) is a difficult to express protein
of Mycobacterium tuberculosis that elicits strong
and specific delayed type hypersensitivity reac-
tions in humans infected with M. tuberculosis.
Therefore DPPD is a molecule that can improve
the specificity of the tuberculin skin test, which
is widely used as an aid for the diagnosis of tu-
berculosis. However, a pitfall of our initial stud-
ies was that the DPPD molecule used to perform
the skin tests was engineered as fusion mole-
cule with another Mycobacterium protein. This
approach was used because no expression of
DPPD could be achieved either as a single
molecule or as a fusion protein using a variety
of commercially available expression systems.
Here, we report the production and purification
of rDPPD using a synthetic gene engineered to
contain E. coli codon bias. The gene was cloned
into pET14b expression vector, which was sub-
sequently used to transform Rosetta 2(DE3)
pLysS or BL-21(DE3)pLysS host cells. The re-
combinant protein was over-expressed after in-
duction with IPTG and its purification was easily
achieved at levels of 5 – 10 mg/l of bacterial
broth cultures. The purified protein was con-
firmed to be DPPD by Mass Spectroscopy se-
quencing analysis. Moreover, purified rDPPD
stimulated peripheral blood mononuclear cells
of PPD positive blood donors to produce high
levels of IFN-γ, thus confirming that this mole-
cule is biologically active. Because of the DPPD
gene is restricted to the tuberculosis-complex
organisms of Mycobacterium genus, this highly
purified molecule should be useful for the iden-
tification of individuals sensitized with tubercle
Keywords: Mycobacterium tuberculosis; DPPD;
Codon Bias
Over-expression of recombinant proteins in Esche-
richia coli host cells followed by purification in affinity
resins has become a routine and useful procedure to
produce a variety of antigenic molecules. However, not
seldom and for not entirely known reasons, the bacterial
host cells fail to properly express the recombinant pro-
teins coded for by the genes cloned into a number of
different expression vectors. Several possibilities have
been raised to explain this difficult-to-express proteins
puzzle. These include protein toxicity to E. coli, plasmid
or protein instability, inefficient transcription or transla-
tion, inefficient post-translational modification, and pre-
sence in the cloned gene of inadequate or non-used co-
don sequences by the E. coli host cells [1,2].
A typical example of difficult-to-express protein is
DPPD, a Mycobacterium tuberculosis molecule that we
have described several years ago [3,4]. The name DPPD
represents the single letter code of the first four amino
acids of the N-terminus sequence of the mature form of
the protein (aspartic acid, proline, proline, aspartic acid).
DPPD is a small protein composed of 84 amino acids
and is a major component of the complex protein mix-
ture present in purified protein derivative (PPD), which
is the antigenic preparation used in the skin test per-
formed in humans and cattle for the diagnosis of tuber-
culosis [5-9]. Therefore, DPPD is interesting candidate
molecule to be used as a purified antigen for the diagno-
sis of tuberculosis. However, expression of recombinant
DPPD in E. coli host cells either as a single molecule or
as a fusion protein, using an exhaustive list of commer-
cially available expression systems for this host cells,
consistently failed. Moreover, rDPPD could not be ex-
pressed in other cell systems such as the yeast Pichia
pastoris and Streptococcus lividans. In addition, at-
tempts to produce a synthetic DPPD polypeptide through
several protein manufacturing services were also unsuc-
*Financial support: This work was supported by the National Institutes
of Health
rant R01AI076425.
S. S. Kashino et al. / Open Journal of Immunology 1 (2011) 1-7
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJI/
cessful. Partial success to express rDPPD as a single
molecule was achieved using Mycobacterium smegmatis
as host cells transformed with the plasmid pSMT3 [10].
Unfortunately, the yields ofpurified protein obtained with
this system was extremely low (~500 μg/liter of bacterial
culture medium), which precludes the production of the
protein for biological or clinical studies. However, be-
cause in this system both the host cells and the cloned
gene belong to the same bacterial genus (Mycobacte-
rium), these studies suggested that shared preferential
codon usage between the two species facilitated the ex-
pression of rDPPD in M. smegmatis.
In the present work we tested this possibility. A syn-
thetic DPPD gene containing a sequence designed to
have optimized E. coli preferential codons was obtained
and cloned into pET14b. Transformation of E .coli host
cells with this plasmid followed by induction with IPTG
resulted in consistent and successful over-expression
rDPPD, which could be purified by affinity chromatog-
raphy (Ni-NTA resin) yielding 5 - 10 mg of purified re-
combinant protein per liter of bacterial culture.
2.1. DPPD Codon Optimization and
Gene Subcloning
The codon optimization for maximum expression in E.
coli was performed using iteratively sampling from a
codon usage table to find a low free energy solution,
which typically results in decreased secondary structure
of the mRNA. The DPPD optimized sequence was syn-
thesized at Blue Heron (https://www.blueheronbio.com)
using a proprietary technology which allows 100% ac-
curacy. An Nde I and a Bam HI restriction enzyme se-
quences flanking the 5’ and 3’ ends respectively were
incorporated in the DPPD gene sequence. The synthe-
sized DNA was cloned into pUCminusMCS (Blue Heron)
followed by sequencing, which confirmed the designed
optimized sequence. DPPD synthetic gene was then sub-
cloned into the pET14b expression vector (Novagen
EMD Chemicals, Gibbstown, NJ) using the two restric-
tion enzymes. Successful subclones were confirmed by
PCR using Taq Plus DNA polymerase (Invitrogen) and
DNA sequencing.
2.2. rDPPD Protein Expression and
Plasmid pET14b harboring the DPPD optimized gene
was used to transform Escherichia coli BL21(DE3)
pLysS or Rosetta 2 (DE3)pLysS (Novagen) expression
hosts. 100 ml LB cultures of IPTG-induced E. coli were
processed under native or denaturing conditions followed
by affinity chromatography using nickel-nitrilotriacetic
acid (Ni-NTA) agarose matrix (Qiagen, Valencia, CA).
Purified rDPPD was analyzed by SDS- PAGE and con-
centration was determined by BCA (Pierce Thermo Sci-
entific, Waltham, MA).
2.3. Western Blot
Purified recombinant DPPD protein was electropho-
resed on SDS-PAGE (4% - 20% gradient, BioRad, Her-
cules, CA) under reducing conditions and transferred to
a PVDF membrane (Immobilon-P, Millipore, Billerica,
MA). The blot was blocked for one hour at room tem-
perature with Tris-HCl buffer pH 7.4 containing 0.1%
Tween 20 and 1% BSA, andsubsequently incubated with
HRP-labeled anti-His tag monoclonal antibody (Invitro-
gen, Carlsbad, CA). Bound conjugate was detected by
using the LumiGlo chemiluminescent system (Cell Sig-
naling Technology, Danvers, MA), and visualized by ex-
posing the membrane to an autoradiography film (Kodak
BioMax, Rochester, NY).
2.4. Human Interferon-Gamma Response
Blood samples (10 ml) were collected with informed
consent from 6 healthy, confirmed PPD positive donors.
Human peripheral blood mononuclear cells (PBMC) were
isolated by gradient centrifugation (Histopaque, Sigma-
Aldrich, St. Louis, MO). 2 × 105 cells PBMC in com-
plete RPMI supplemented with 10% human AB serum
were incubated (37˚C, 5% CO2) with different concen-
trations of rDPPD (0.4 to 10 μg/ml) or PPD (2.5 μg/ml
to 10 μg/ml); PHA (1/500) or medium alone was in-
cluded as positive and negative controls, respectively.
Supernatants were collected after 72 h of culture and as-
sayed for IFN-γ content using commercial capture and
detection mAb pairs for sandwich ELISA (BD Biosci-
ences, Rockville, MD).
3.1. Codon Optimization
The M. tuberculosis DPPD full length gene (Rv0061)
codes for a typical secreted protein, which includes a
signal peptide sequence of 39 amino acids followed by
the signal peptidase recognition sequence Ala-Ser-Ala
(Figure 1). Because in our original studies [3,4,10] we
found that M. tuberculosis produces only the mature
sequence of the protein we opted to evaluate the codon
optimization designed to include only this form of the
molecule. To facilitate expression an initiation ATG
codon was added to 5’ end of the gene sequence. Figure
2 depicts the DPPD optimized gene sequence compared
to that of M. tuberculosis.
S. S. Kashino et al. / Open Journal of Immunology 1 (2011) 1-7
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJI/
Figure 1. Deduced amino acid sequence of the protein coded
by Mycobacterium tuberculosis Rv0061. The deduced amino
acid sequence of the 112 residues of the protein, reveals that
the full length gene codes for a typical secretory protein, which
includes a signal peptide sequence (illustrated in red letters),
the signal peptidase recognition sequence ASA (illustrated in
green, bold, italic letters) and the mature sequence of protein
(DPPD), which is depicted in black bold letters.
3.2. Protein Expression and Purification
The DNA fragment containing the synthetic optimized
DPPD sequence was cut from pUCminusMCS and sub
-cloned into pET-14b vector. This expression vector con-
tains a Histidine tag (His-tag) sequence before the Nde I
cloning site thus generating a recombinant protein con-
taining a sequence of six His residues at the N-terminus
to facilitate its purification by affinity binding to a
Ni-NTA agarose matrix. The expression vector was used
to transform Rosetta 2(DE3)pLysS or BL21 (DE3)pLysS
host cells which were then induced with IPTG. Recom-
binant protein expression was assessed by SDS-PAGE
with Coomassie blue staining and is illustrated in Figure
3A (lanes 1 and 2 respectively). Over- expressed pro-
teins bands of MW ~12 kDa and ~25 kDa can be seen in
the lane corresponding to the IPTG induced culture. No
such strongly stained bands are seen in the lane corre-
sponding to the non-induced culture (lane 1).
The recombinant protein was purified as soluble pro-
tein from BL21(DE3)pLysS or from the inclusion bodies
in Rosetta 2(DE3)pLysS using affinity chromatography.
Yields of purification ranged from 5-10mg of purified
protein per liter of induced culture. Figure 3 (lane 3B)
shows the purified recombinant protein. Two major band
aggregates are seen at a positions matching the over-
expressed protein seen in the induce cultures (Figure 3A,
lane 2). The lower bands (MW ~8 - 15 kDa) are within
the range of the predicted MW of the native mature form
of DPPD (9.02 kDa). This pattern of migration is usually
observed with proteins with high content in proline
residues [11-14] and is consistent with our former ob-
servations for both native and recombinant molecules
[4,10]. In addition, three other bands of higher MW (~20 -
30 kDa) are also clearly seen. At first, many of these
Figure 2. DPPD DNA sequence with E. coli optimized codon usage. Codon optimization for
protein expression was done using the Blue Heron Expression Optimization tool which mini-
mizes secondary mRNA structure to reduce translational impediments. Optimized DPPD se-
quence is displayed in bold (upper sequence). Note that an Nde I and a Bam HI restriction en-
zyme sequences (underline sequences) flanking the 5’ and 3’ ends respectively were incorpo-
rated in the optimized sequence. For comparison purposes the Rv0061 gene sequence (coding
the mature form of the protein only) is also shown (lower sequence).
S. S. Kashino et al. / Open Journal of Immunology 1 (2011) 1-7
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJI/
Figure 3. Expression of recombinant DPPD in E.
coli. Recombinant DPPD was expressed in E.
coli with six His-tag amino terminal residues
and the protein was purified by affinity chroma-
tography using Ni-NTA agarose matrix. Coo-
massie blue stained SDS/4-20% polyacrylamide
gradient gel of 5μg of purified recombinant
DPPD (lane 1). MWM, molecular weight mark-
ers; numbers on left are the MW of the markers
in kDa.
bands would be assumed to be contaminants. However,
because the mature DPPD contains four cysteine resi-
dues it is also possible that the extra bands seen in the
gel are indeed polymers (or aggregates) of the single
molecule. Although the gel electrophoresis illustrating
the purity of DPPD ran under reducing conditions, it is
possible that reduction was not complete, which results
in several bands of the same molecule been displayed in
the gel.
To test this hypothesis we performed Western blot
analysis carried out under reducing and non-reducing
conditions. Because rDPPD was engineered to contain a
tag of six histidines the identification of the molecule
was performed using an anti-His tag IgG mAb. Figure 4
confirms that this monoclonal antibody recognized the
over-expressed protein. Under reducing conditions (Fig-
ure 4, Lane R) the Western blot confirmed that the two
groups of molecules (~8 - 15 kDa and ~20 - 30 kDa)
were strongly reactive with the anti-His tag mAb, thus
substantiating that these bands are indeed the recombi-
nant DPPD. In addition, the Western performed under
non-reducing conditions (Figure 4, lane NR) clearly
shows that DPPD is present as variety of polymeric mole-
Figure 4. Western blot analyses of recombinant
DPPD expressed by E. coli. Purified rDPPD was
initially subjected to SDS-PAGE (gradient gel 4 -
20) performed under reducing (R) and non-reducing
(NR) conditions. Protein was transferred to nitro-
cellulose membrane and the presence of the recom-
binant molecule was identified using a mouse anti-
His-tag (C-terminus) monoclonal antibody. Reactiv-
ity was detected with peroxidase labeled goat anti
mouse immunoglobulin and developed using ECL
chemo luminescent reagent (Western blot detection
system, Amershan Biologicals, Upsala Swe- den).
Numbers on the left side indicate the molecular
weights of the markers.
cules. Interesting no bands of molecules with MW
smaller than 15 kDa was seen under these conditions.
These results indicate that strong polymerization of the
molecule occurs through its cysteine residues and no
monomeric forms are present in the purified preparation.
Finally, no bands were seen in the blots probed with
control IgG antibody (not shown).
To categorically define that the purified recombinant
protein was indeed DPPD, amino acid sequence was
performed by mass spectroscopy. The recombinant pro-
tein was run on a 4% - 20% gradient gel and the single
bands seen in the Coomassie Blue stained gel were ex-
cised and in gel tryptic digested followed by LC-MS/MS
analysis for identification. rDPPD protein sequence was
identified in bands with high confidence (XCORR:
4.7594) through a single tryptic digested peptide present
within the amino acid sequence of DPPD. The sequence
had 100% identity with the deduced amino sequence of
the DPPD gene at the residues 19 - 35 (Figure 5).
Taken together, these results clearly point that rDPPD
can be over-expressed in Rosetta 2 or BL21 host cells
S. S. Kashino et al. / Open Journal of Immunology 1 (2011) 1-7
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Figure 5. DPPD peptide sequence identified by mass spec-
troscopy in purified recombinant protein. The peptide sequence
and positioning within the peptide donor protein (DPPD) is
highlighted in red/bold/underline. The trypsin cleavage sites
(right side of the amino acids R and K) that generated the pep-
tide are illustrated above the molecule.
transformed with pET-14b containing the DPPD gene
with optimized sequence for E. coli.
3.3. Biological Validation of Purified rDPPD
One important requirement to validate a microbial
molecule as a diagnostic tool or as a vaccine candidate is
that the immune response of a host sensitized or infected
with the microbe donor of that protein recognizes that
molecule. Our former studies using rDPPD expressed as
fusion protein have confirmed that guinea pigs and hu-
mans sensitized or infected with M. tuberculosis develop
specific T cell response to DPPD. Therefore, it became
important to verify that the rDPPD molecule expressed
and purified in the present work would also be recog-
nized by T cells from M. tuberculosis sensitized indi-
viduals. To test this requirement, PBMC were obtained
from six healthy volunteers who were known to be re-
sponders to purified protein derivative (PPD) of tubercu-
lin, the antigen that is used in the human skin test for the
diagnosis of tuberculosis. Recognition of rDPPD was
tested by antigen-induced production of IFN-γ by the
donors’ PBMC. As it can be seen in Figure 6, rDPPD
stimulated the PBMC of all six tuberculin sensitive do-
nors to produce high levels of IFN-γ. No response was
observed with PBMC obtained from three PPD non-
responder controls (not shown). Due to the limited
number of individuals analyzed these results cannot at
this point be correlated with clinical validation of rDPPD
as a tool to be used in the diagnosis of tuberculosis.
However, because rDPPD isreadily recognized by
PBMC of tuberculosis sensitized individuals the results
clearly indicate that the newly expressed and purified
recombinant molecule is biologically active.
I early studies we showed that a recombinant fusion
molecule composed of rRa12-DPPD elicited delayed
type hypersensitivity in humans comparable to that elic-
ited by standard PPD antigen [3]. Unfortunately, this
fusion protein contains a 14 kDa polypeptide from M.
tuberculosis which, in contrast to DPPD, is broadly dis-
Figure 6. Recognition of purified recombinant DPPD by hu-
man PBMC. IFN-γ production by PBMC from PPD positive
healthy donors following stimulation for 72 h with medium,
rDPPD (10 μg/ml) or PPD (10 μg/ml) was measured by sand-
wich ELISA in the culture supernatants. Bars represent the SD
of the means calculated from the results of triplicate cultures.
tributed among the Mycobacterium genus. If on one
hand we were successful to generate a purified recom-
binant molecule, on the other hand it introduced an un-
desired property to the rDPPD i.e. , a fusion protein that
is no longer specific for M. tuberculosis. However, this
“homemade” fusion protein expression system [15] was
the only procedure that we found to be successful to
produce rDPPD. A variety of commercially available
systems that uses E. coli as host cells consistently failed
to express rDPPD. These included various vectors such
pET, pQ30 (Qiagen), pThioHis (Invitrogen), and pGEX-
2T (GE Healthcare, Piscataway, New Jersey). Host E.
coli cells tested included BL21(DE3), BL21(DE3)pLysS,
and Rosetta 2(DE)pLysS (Novagen). In addition, rDPPD
could not be expressed using other host cells such as the
yeast Pichia pastoris or Streptococcus lividans (unpub-
lished observations).
However, as mentioned before, low levels of rDPPD
could be expressed and purified by using the Mycobac-
terium expression vector pSMT3 and Mycobacterium
smegmatis as host cell [10]. This observation prompted
us to hypothesize that preferential codon usage could
have been the condition that facilitated the synthesis of a
M. tuberculosis protein in a Mycobacterium host cell.
Here, we tested this possibility using a standard expres-
sion vector (pET14b) and standard E. coli host cells
(Rosetta 2(DE3)pLysS or BL21(DE3)pLysS), which are
a well known systems designed for high levels of protein
production. The technological advent of achieving ro-
bust and automated synthesis of large DNA molecules
permitted us to produce a DPPD gene containing a se-
quence designed to match the preferential codon usage
of E. coli instead of that of Mycobacterium. The E. coli
S. S. Kashino et al. / Open Journal of Immunology 1 (2011) 1-7
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJI/
optimized DPPD gene was synthesized by BlueHeron
Biotechnology, Seattle WA. Blue Heron uses proprietary
codon utilization databank, geneassembly instruments,
and other technologies to accurately and rapidly engi-
neer and assemble oligonucleotides into full-length con-
structs. Using BL21 or Rosetta 2 E. coli transformed
with pET14b containing the optimized DPPD gene we
were able to successfully express and purify rDPPD in
yields never before achieved. Approximately 5 - 10 mg
of purified recombinant was consistently obtained per
liter of bacterial culture.
The deduced MW of mature DPPD molecule (9,022.9
Da) does not agree with the molecular mass of ~12 kDa
of the major band of the purified molecule estimated by
SDS-PAGE. However, this same pattern of migration
was observed with the native molecule when we first
discovered DPPD [4]. This phenomenon of abnormal gel
migration has been described for several proteins that
have an unusually high proline content and a low iso-
electric point caused by high contents of aspartic and glu-
tamic acid residues [12-14]. In general, the classical SDS-
PAGE method often overestimates molecular weights of
molecules if the proline content is >10% in a given pro-
tein [11]. The general consensus is that this altered mi-
gration pattern is caused by the amino acid composition,
and not post-translational modification [14]. Coinciden-
tally DPPD fits all these predictive parameters. Out of
the 84 amino acids that compose the mature form of the
molecule, 16 (19%) are prolines, 7 (8.3%) are aspartic
acid and 3 (3.6%) are glutamic acid. Moreover, the theo-
retical pI of the mature DPPD protein is 4.2, which was
obtained using the ExPASy Proteomics algorithm of The
Swiss Institute of Informatics (http://au.exp asy.org/tools/
protparam.html). Therefore, these unique molecular cha-
racteristics of the mature rDPPD are in consonance with
its pattern of migration observed in the PAGE analysis.
Importantly, the 12 kDa band seen in the PAGE was
confirmed to be DPPD by mass spectroscopy.
Also interesting was the confirmation that the purified
recombinant DPPD was readily recognized by the PBMC
obtained from tuberculosis sensitized healthy individuals.
These observations point to the potential use of this sin-
gle and defined molecule as a potential reagent for the
tuberculin skin test. Alternatively, rDPPD can be also an
interesting molecule to be tested as component of the
recently developed whole blood IFN-γ release assay for
the diagnosis of tuberculosis. As mentioned before, this
molecule is unique to members of the M. tuberculosis
complex only, therefore an attractive specific antigen for
test development.
Finally, it is important to emphasize that the procedure
described in this manuscript to achieve workable con-
centrations of rDPPD per liter of bacterial culture, uses
conventional standard operating procedures for produc-
tion of recombinant proteins. Therefore, if rDPPD, in
future experiments, proves to be useful for the diagnosis
of tuberculosis, no hurdles should exist to upscalethe
production of this molecule under GLP or GMP condi-
tions for clinical use.
DPPD, a difficult to express protein of Mycobacte-
rium tuberculosis.
None of the authors has any financial conflict of in-
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