Biotechnology Letters 26: 7578, 2004.

2004 Kluwer Academic Publishers. Printed in the Netherlands.

75

Expression of ornithine decarboxylase of Coccidioides immitis in three

Escherichia coli strains carrying the lambda DE3 lysogen and an E. coli
EWH319 strain odc null mutant
Miguel Angel Pantoja-Hernandez1 , Claudia Ivonne Muoz-Sanchez1 , Ramon Gerardo Guevara-Gonzalez1 , Enrique Botello-Alvarez1 , Mario Martn Gonzalez-Chavira2 ,
Irineo Torres-Pacheco2 & Lorenzo Guevara-Olvera1,
1 Instituto

Tecnologico de Celaya, Departamento de Ingenieraa Bioqumica, Ave. Tecnologico y A. Garcia-Cubas,

S/N, Colonia FOVISSSTE, Apartado postal 57, Celaya, Guanajuato, Mexico
2 Instituto Nacional de Investigaciones Forestales Agrcolas y Pecuarias (INIFAP), Unidad de Biotecnologa del
Bajo, Carretera Celaya-San Miguel de Allende Km 6.5, Apartado Postal 112, Celaya, Gto, Mexico
Author for correspondence (Fax: +52-461-6117979; E-mail: lorenzogo@yahoo.com)
Received 11 September 2003; Revisions requested 22 September 2003; Revisions received 4 November 2003; Accepted 5 November 2003

Key words: Coccidioides immitis, Escherichia coli, gene function, lambda DE3 lysogen, protein expression

Abstract
Ornithine decarboxylase from respiratory fungal pathogen, Coccidioides immitis, cloned in the pETCiODC plasmid under control of T7lac promoter, was produced in E. coli BL21(DE3), BL21(DE3)pLysS, BLR(DE3) and
EWH319 transformant strains. E. coli BL21(DE3)pLysS-pETCiODC expressed the highest specific activity of
ODC, suggesting that this strain could be successfully used for protein structure and drug testing studies.

Introduction
Coccidioides immitis is a desert soil fungus found in
the Southwest of the United States, North of Mexico, and other parts of the Western hemisphere. It is a
human pathogen that by inhalation infects immunologically normal as well as immunocompromised hosts
(Galgiani 1993). Several studies on C. immitis have
emphasized the identification of novel molecular targets of potential antifungal agents (Yu et al. 1999,
Cole et al. 1994). Polyamines play a key role in
the differentiation of dimorphic fungi, between yeast
and mycelial phases of growth (Guevara-Olvera et al.
1997). The synthesis of polyamines in this group
of microorganisms typically involves production of
ornithine from arginine in the presence of arginase,
followed by the formation of putrescine catalized by
ornithine decarboxylase (ODC). Addition of ODC inhibitors to yeast cultures blocks the yeast-to-mycelium
transition (Pfaller et al. 1988, Guevara-Olvera et al.
1997). Recently, the ODC gene of C. immitis was

cloned and it has been demonstrated that its activity

is fundamental for the parasitic cycle (Guevara-Olvera
et al. 2000).
Expression of heterologous genes in microbial systems depends on its components, including the protein synthetic machinery (Smith 1996). Heterologous
genes are commonly expressed at high levels, as much
as 40% (w/w) of the total protein in several systems
(Alldred et al. 1992, Smith 1996). The pET DNA
vector cloning system, containing a hybrid T7lac promoter (Dubendorff & Studier 1991), is the most commonly used and probably the most renowned bacterial
expression system available (Viaplana et al. 1997).
The present paper reports the ODC expression of
C. immitis with and without IPTG using pET28b expression vector, and three E. coli strains lambda DE3
lysogen and an E. coli EWH319 strain odc null
mutant.

76
Materials and methods
Bacterial strains and plasmid
E. coli strains lambda lysogen BL21(DE3), BL21
(DE3)pLysS and BLR(DE3) and an E. coli strain
EWH319 odc null mutant (ATCC 33232) (Hafner
et al. 1979) were transformed with the plasmid pETCiODC, containing the ODC gene from C. immitis
under control of the T7lac promoter and the kanamycin resistance gene. pET-CiODC plasmid has been
reported elsewhere (Guevara-Olvera et al. 2000).
Analysis of ODC expression in E. coli strains lambda
lysogen by SDS-PAGE separations
Cultures incubated with shaking at 37 C presenting OD600 = 0.6 (about 3 h) were used for ODC
expression with or without 0.4 mM IPTG. Samples,
3 ml, taken at different times (0, 1, 2, and 3 h)
during induction were ice-cooled. Cells were collected, resuspended in loading buffer and boiled for
5 min. Volumes corresponding to the same protein
amount (1520 g) determined by the Bio-Rad protein assay were loaded in SDS-PAGE and visualized
by Coomassie Blue staining. The ODC accumulating
in inclusion bodies was dissolved in 6 M urea and
isolated by nickel-affinity chromatography, separated
by SDS-PAGE and electrotransferred to hydrophobic
polyvinylidene difluoride (PVDF) membrane, excised
and digested with Lys-C in preparation for amino acid
sequence analysis (Yu et al. 1999).
ODC specific activity expressed in E. coli strains
lambda lysogen
ODC dissolved in 6 M urea and isolated by nickelaffinity chromatography was dialysed on PBS buffer
(pH 7.3) using a membrane with a cut-off of 20 kDa
for gradual removal of the urea, salt and imidazole
present under elution conditions. After dialysis, the
ODC was concentrated by sprinkling solid polyethylene glycol (20 000 MW) on dialysis tubing with an
exclusion limit of 6000 MW until reaching the desired
volume. The protein was resuspended in PEDP buffer
and used to assay for ODC activity as described elsewhere (Fonzi & Shyperd 1985). The radiometric assay
which detects 14 CO2 evolved from [1-14C]ornithine
(Sigma-Aldrich, St. Louis, MO) was conducted to
analyze 3 separate preparations of E. coli transformants lambda lysogen. The protein concentration of

Fig. 1. SDS-PAGE analysis of ODC overexpression by E. coli

strains lambda lysogen. Lane 1, molecular weight marker.
Lanes 29 show electrophoresis separations of cytosolic proteins
(20 g) from E. coli BL21(DE3) (lanes 25), BL21(DE3) pLysS
(lanes 6, 7) and BLR(DE3) (lanes 8, 9) transformed with either
pET28b plasmid alone (lanes 2, 3), or pET-CiODC plasmid
(lanes 49) without IPTG induction (lanes 2, 4, 6, and 8) and with
IPTG induction (lanes 3, 5, 7 and 9).

Fig. 2. SDS-PAGE analysis of ODC overexpression. Lines 1

and 10, molecular weight marker. Lanes 29 show electrophoresis separations of cytosolic proteins (15 g) from E. coli
strain BL21(DE3)pLysS transformed with the pET-CiODC plasmid, without IPTG induction (lanes 25) and with IPTG induction
(lanes 69) after 0, 1, 2 and 3 h, respectively.

aliquots of each sample was determined by the BioRad Protein Assay. The ODC specific activity was
recorded as pmol 14 CO2 released min1 mg protein.
ODC specific activity expressed in an E. coli strain
odc null mutant
E. coli transformat cells were grown in M9 minimal
medium containing 50 g kanamycin/ml, washed with
PEDP buffer and the cell pellet was then homogenized
by sonication in the same buffer. The homogenate was
centrifuged at 17 600 g, 4 C for 20 min and the supernatant was used to assay for ODC activity as above.
Confirmation of ODC specific activity in E. coli strain
EWH319-pETCiODC was performed using an inhibitor 1,4-diamino-2-butanone (DAB) which specifically
blocks eukaryotic ODC activity. DAB (Sigma) at
110 mM was added to the cells homogenates.

77
Table 1. ODC activity produced by E. coli strains lambda lysogen and E. coli EWH319
transformed with the pET-CiODC expression plasmid.
Parental strain

Plasmid

1,4-Diamino-2butanone (mM)

Specific
activitya

Inhibition (%)

BL21(DE3)
BL21(DE3)pLysS
BLR(DE3)
EWH319

pET-CiODC

0
0
0
0
0
0.1
1
5
10

2094 73
2527 56
1974 67
6.5 1.3
1149 25
999 56
819 39
391 28
131 26

13
29
34
88

pET28b
pET-CiODC

a Expressed as pmol 14 CO released min1 mg protein.

2

Results and discussion

Expression of ODC from C. immitis in E. coli strains
lambda lysogen
Total cytosolic preparation derived from transformant cells either induced or not with IPTG during
growth are shown in Figure 1. A prominent Coomassie
Blue-stained band with a molecular mass of 52 kDa
was visible after SDS-PAGE separation (lanes 4
9). Over-expression induced by IPTG considerably
increased the relative amount of protein produced
by E. coli strain BL21(DE3)-pETCiODC transformant cells (lane 5). The nature of these differences
on expression with and without IPTG is open to
be further characterized in this strain. On the other
hand, E. coli strain BL21(DE3)pLysS-pETCiODC
and BLR(DE3)-pETCiODC transformant cells produced similar amount of ODC with and without IPTG
(lanes 6 and 7; lanes 8 and 9), respectively. These
results confirm that over-expression of heterologous
proteins without IPTG is possible using pET28b expression vector and as host E. coli strains carrying
lambda DE3 lysogen (Dubendorff & Studier 1991).
The effect of IPTG on the production of ODC by
E. coli strain BL21(DE3)pLysS was analyzed by SDSPAGE of cell extracts at 0, 1, 2 and 3 h. In Figure 2,
kinetics for both conditions with or without IPTG are
shown, revealing that ODC is already detected at time
zero (inoculum of 3 h) (lanes 2 and 6) and its amount
increases in a time-dependent fashion in the absence
(lanes 35) or the presence of IPTG (lanes 79). These
results suggest that T7 RNA polymerase encoded by
a single copy of lambda DE3 lysogen expressed as
a constitutive gene into the genome of E. coli strains

(Dubendorff & Studier 1991), supports the expression

of targeted genes under T7lac promoter contained into
pET28b expression vector.
The ODC produced into inclusion bodies by
E. coli strain BL21(DE3)pLysS-pETCiODC was LysC-digested (Yu et al. 1999), separated by HPLC, and
a selected peptide purified to homogeneity was subjected to N -terminal sequence analysis. The obtained
sequence (K)AVEDARFVFDQASDI, which matches
the aa sequence of the translated ODC gene from
C. immitis (aa 242-257, accession number A179245),
demonstrated the ODC overexpression.
ODC specific activity in E. coli strains lambda
lysogen and E. coli strain EWH319
ODC specific activity expressed in three E. coli strains
lambda lysogen is shown in Table 1. In E. coli
strain BL21(DE3)pLysS-pETCiODC, the ODC specific activity was 18 and 22% higher than ODC
expressed in BL21(DE3)-pETCiODC and BLR(DE3)pETCiODC transformant cells. The genetic background in E. coli strains lambda lysogen is essentially
similar and the ODC purification and refolding was
performed using same protocol, probably the differences in activity could be attained to the wild type
ODC gene harbored in the genome of E. coli strains
lambda lysogen.
E. coli strain EWH319-pETCiODC transformant
cells expressed ODC specific activity recorded as
pmol 14 CO2 released min1 mg protein (Table 1).
To evaluate whether the radiometric assay detected
14 CO which evolved from [1-14 C]ornithine as a result
2
of ODC activity, a competitive and specific inhibitor
of the enzyme was added at different concentrations

78
to the reaction mixture. Approx. 90% inhibition of
enzyme activity was recorded when 10 mM DAB was
added, confirming that the ODC gene from C. immitis
encodes a protein that catalyzes the decarboxylation of
ornithine.
In conclusion. The isolation of ornithine decarboxylase from C. immitis expressed under T7lac
promoter by three E. coli strains carrying lambda
(DE3) lysogen with and without IPTG and ODC specific activity in an E. coli odc null mutant is reported.
For the economic feasibility to get proteins in high
quantity and quality for biophysical studies as crystallography and spectroscopy, overexpression systems as
pET28b and E. coli (DE3) strains are useful. Additionally, E. coli EWH319 strain from which the ornithine
decarboxylase is deleted could be used to screen inhibitors for eukaryotic ODCs. Therefore, the ODC
production from this fungus in E. coli for structural
and functional studies may provide some useful information to detect potential targets for rational design
of antifungal drugs.

Acknowledgements
We thank Dr Rita Miranda Lpez for helpful discussion and English correction. Financial support by
COSNET Grant No. 566.01-P.

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