Successful expression and purification of dppd, using a codon optimized synthetic gene

doi:10.4236/oji.2011.11001

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Vol.1, No.1, 1-7 (2011) Open Journal of Immunology

doi:10.4236/oji.2011.11001

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.

ABSTRACT

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

bacilli.

Keywords: Mycobacterium tuberculosis; DPPD;

Codon Bias

1. INTRODUCTION

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

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. MATERIAL AND METHODS

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

Purification

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. RESULTS

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

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

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

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.

4. DISCUSSION

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

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.

5. ABBREVIATIONS

DPPD, a difficult to express protein of Mycobacte-

rium tuberculosis.

6. CONFLICT OF INTEREST

None of the authors has any financial conflict of in-

terest.

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