Journal of Biotechnology 127 (2006) 109–114

Scale-up of recombinant Opc protein production in

Escherichia coli for a meningococcal vaccine
Raúl Espinosa Pérez b , Alexis Musacchio Lasa a,∗ , Ricardo Silva Rodrı́guez a ,
Evelin Caballero Menéndez a , José Garcı́a Suárez b , Héctor Dı́az Balaguer b
a División de Vacunas, Centro de Ingenierı́a Genética y Biotecnologı́a, Ave 31 e/158 y 190, Apdo. Postal 6162,
Cubanacán, Playa, La Habana 10600, Cuba
b División de Desarrollo Biofarmacéutico, Centro de Ingenierı́a Genética y Biotecnologı́a, Ave 31 e/ 158 y 190,
Apdo. Postal 6162, Cubanacán, Playa, La Habana 10600, Cuba

Received 20 March 2006; received in revised form 2 June 2006; accepted 14 June 2006

Abstract

Opc is an outer membrane protein from Neisseria meningitidis present in meningococcal vaccine preparations. The opc gene,
codifying for this protein, was cloned in to Escherichia coli and the Opc protein was expressed under the control of a tryptophan
promoter. The recombinant strain was grown in batch cultures. Opc was expressed as inclusion bodies at about 32% of the total
cellular protein. We examined the scale-up culture conditions for the production of the recombinant Opc. The scale-up process
was performed from 1.5 l to 50 l culture, using first, the constant power per unit of volume (P/V) as main scaling criteria, and
then the oxygen mass transfer coefficient (KL a) scaling criteria to adjust the optimal aeration conditions. A final productivity of
52 mg l−1 h−1 was obtained at the 50 l culture scale compared with the 49 mg l−1 h−1 productivity at 1.5 l laboratory scale.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Neisseria meningitides; Opc; Recombinant protein; Scale-up

1. Introduction sustained immune response in infants and do not elicit

Neisseria meningitidis is a human pathogen respon-
sible for a large number of clinical cases of bacterial
meningitis worldwide (Peltola, 1983). Current vac-
cines consist of the group A, C, W-135 and Y capsular
polysaccharides (cPs). These vaccines fail to invoke a
∗ Corresponding author. Tel.: +53 7 2716022; fax: +53 7 2714764.
E-mail address: alexis.musacchio@cigb.edu.cu (A.M. Lasa).
memory response (Reingold et al., 1985). Development
of safe and effective vaccines based on the serogroup
B capsular polysaccharide is complicated by its poor
immunogenicity and the existence of identical struc-
tures in the human host. Therefore, vaccines based
on meningococcal outer membrane proteins (OMPs)
against serogroup B meningococci are currently under
investigation. The Opc protein is present in the OMP-
based vaccines tested and it is partially responsible

0168-1656/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2006.06.005
110 R.E. Pérez et al. / Journal of Biotechnology 127 (2006) 109–114
for the protection they confer (Fredriksen et al., 1991;
Sierra et al., 1991). The Opc protein is a meningococ-
cal antigenically conserved OMP, which is expressed
by a large subset of strains. It is heat-modifiable,
trimeric and has a basic isoelectric point (Achtman
et al., 1988). The molecular mass of the monomer is
28 kDa. The Opc protein is highly immunogenic in
human (Rosenqvist et al., 1993). Its coding gene, opc,
was isolated, cloned and expressed in Escherichia coli
(Guillen et al., 1995). These facts have made the Opc
protein to be considered as a vaccine candidate against
meningococci.
In this work we studied and established the fermen-
tation process of a particular Opc expression clone and
for which we selected like objective function the volu-
metric production when changing of scale.
2. Materials and methods
2.1. E. coli strain and expression plasmid
E. coli K12 GC366 (Sambrook et al., 1989) strain
was transformed with the plasmid pM-107 (Musacchio
et al., 1997) which contains the entire opc gene encod-
ing for the Opc protein under the control of the trypto-
phan (trp) promoter (Novoa et al., 1995).
2.2. Growth conditions
The recombinant strain was grown in 100 ml LB
medium (Sambrook et al., 1989), overnight at 37 ◦ C
and 150 rpm, supplemented with 100 ␮g tryptophan
ml−1 (Merck, Germany) and 50 ␮g kanamycin ml−1
(Sigma Co. Ltd., USA), in shake flasks 500 ml (New
Brunswick Scientific Co., USA).
For growth in fermenters the minimal growth
medium (MM) (Sambrook et al., 1989) was supple-
mented with 10 g tryptone l−1 , 10 g yeast extract l−1 ,
10 g glucose l−1 , 0.015 g CaCl2 ·2H2 O l−1 , 0.246 g
MgSO4 ·7H2 O l−1 and 50 ␮g kanamycin ml−1 . This
process was carried out in 1.5 l fermenters (B.H. Maru-
bishi Co. Ltd., Japan) at 37 ◦ C, 350 rpm, aeration rate
1.5 l min−1 and the pH was controlled at 7.0 with 1 M
NaOH or 0.4 M H3 PO4.
The scale-up batch process was optimized in 50 l
fermenters (B.H. Marubishi Co. Ltd., Japan) at 37 ◦ C,
205 rpm, with an aeration rate 50 l min−1 , using combi-
nation of the criteria of scaled constant power per unit
of volume and the oxygen mass transfer coefficient.
2.3. Analysis
The cell dry weight as indicative parameter of cell
growth was measured using the Sartorius MA 30 mois-
ture balance (Sartorius, Germany).
The recombinant Opc expression levels were deter-
mined by densitometry, after denaturing 10% (SDS-
PAGE) of the E. coli whole cells. The protein concen-
tration was determined by the Lowry method.
3. Results and discussion
Several heterologous proteins have been cloned and
expressed in E. coli under the control of the trp pro-
moter. However, very few reports have detailed the
effects of the induction conditions on the kinetics of
growth and protein expression, essential to obtain a
high yield in the production of recombinant proteins
(Iijima et al., 1987; Shimizu et al., 1987; Park and Seo,
1989).
3.1. Influence of the tryptophan and the tryptone
on the growth conditions and expression of Opc
protein
The trp promoter has been widely used for pro-
ducing recombinant proteins (Nakahama et al., 1988;
Shimizu et al., 1991). The repression of the promoter by
tryptophan is incomplete, and the gradual expression
of cloned genes is observed (Kane and Hartley, 1988).
In our case, the combination of tryptone (contains
100 mg l−1 of tryptophan) and yeast extract (contains
85 mg l−1 of tryptophan) as supplements of tryptophan
(185 mg l−1 ) and vitamins in the culture media was suf-
ficient to keep the expression system under control the
first 2 h of growth. At this moment, the expression of
the recombinant Opc protein started to be detected.
3.2. Scale-up conditions for growth and
expression of Opc recombinant protein
The typical schematic fermentation process, for the
production of the recombinant Opc protein at 1.5 l scale
is shown in Fig. 1A. During the first 6 h of growth an
 R.E. Pérez et al. / Journal of Biotechnology 127 (2006) 109–114 111

Fig. 1. Culture kinetics of Escherichia coli GC366 carrying the plasmid pM-107, grown in 1.5 l fermenters (A) and 50 l fermenters (B). The
cells were grown 12 h at 37 ◦ C, 350 rpm, 1.5 l min−1 . The cell dry weight is expressed in g l−1 (䊉). The expression level of the recombinant Opc
protein () is expressed in percent. The production of the recombinant Opc protein () is expressed in mg l−1 . The amount of recombinant
Opc protein produced per liter culture per growth time determined the productivity of the process ().

increase in the optical density (cell dry weight) was obtained. The biomass yield on substrate (Yx/s ) and
observed. The maximum value of the specific growth the product yield on substrate (Yp/s ) values were 0.29 g
rate was determined during the first 2 h of growth biomass/g substrate and 0.042 g product/g substrate,
(μ = 1.6 h−1 ), being stabilized during the rest of the fer- respectively.
mentation process at μ = 0.98 h−1 , when an increase in The scale-up procedure was performed using the
the expression of the recombinant protein was detected. constant power per unit of volume criteria according
The levels of expression for the Opc protein produc- the following equations:
tion were increased through the fermentation process
during 10 h, reaching at this point also the highest pro- (P/V )1.5l = (P/V )50l (1)
ductivity level.
At this scale, 3 g cell dry wt l−1 , 28% level of recom- P = Pt (Pg /Pt )Nfc (2)
binant Opc protein expression, 418 mg 1−1 volumet-
ric production and 49 mg l−1 h−1 productivity, were P = Np ρn3 di5 (3)
112 R.E. Pérez et al. / Journal of Biotechnology 127 (2006) 109–114

Na = Q/ndi3
where P corresponds to the final power (real power),
Pt is the theoretic power, Pg the gassed power, N the
number of impellents, Np the power number, ρ the fluid
density, n the rotation speed of impeller, di the impellent
diameter, Na the aeration number, Q the aeration rate
and fc is the geometric factor. The geometric factor was
calculated as fc = (DH)1/2 /(3di ), where D is the vessel
diameter and H is the static height of broth.
Substituting (2) and (3) in Eq. (1),
n50l = n1.5l (di1.5l /di50l )5/3
×[(V50l /V1.5l )(N1.5l /N50l )(fc1.5l /fc50l )]1/3
To calculate the aeration rate (Q) at 50 l scale cul-
ture, the constant aeration number at both scales was
employed. Then,
Q50l = Q1.5l n50l /n1.5l (di50l /di1.5l )3
The scale-up parameters employed in the 50 l fer-
mentation processes were calculated using the previ-
ous described Eq. (5) and (6). The obtained results
are shown in Table 1. When fermentation pro-
cess at 50 l scale was performed using the above
calculated parameters (rotation speed of impeller
205 rpm and aeration rate 20 l min−1 ), the final pro-
ductivity was 26 mg l−1 h−1 in contrast with the
49 mg l−1 h−1 obtained at the 1.5 l scale. These results
(4) did not correspond with the expected values. To over-
come this problem the aeration rate was recalcu-
lated using the volumetric oxygen transfer coefficient
constant (KL a) scaling criteria (Aiba et al., 1973;
Asenjo and Merchuk, 1994; Perez and Rodriguez,
1997; Quintero, 1990).
A second scale-up criteria to improve the produc-
tivity by increasing the dissolved oxygen in the culture
media, was employed. In this case, the same rotation
speed of impeller in the (P/V) constant criteria was
taken into account, but using the aeration rate from
the constant criteria of KL a. In this case, the volumet-
ric transfer coefficient (KL a) was calculated by trial
and error, fixing an aeration rate value at the 50 l scale
(5) until obtaining the same value KL a in both scales.
This value was calculated using the Bioreactor Design
Software v 2.4, under the van’t Riet equation (van’t
Riet, 1979) for the oxygen mass transfer coefficient
(KL a).
(6) 0.2
KL a = 2 × 10−3 (P/V )0.7 (Q/π/4(D)2 ) (7)
Using Eq. (7), the calculated value for aeration rate was
50 l min−1 .
The production of the recombinant Opc protein at
50 l scale fermenters, using the recalculated parameter
is shown in Fig. 1B. The results obtained were com-
parable with those from the 1.5 l fermentation scale
(Table 1). The expression of recombinant Opc protein
is shown in Fig. 2.

Table 1
Scale-up fermentation parameters for 1.5 l and 50 l fermentation process
Parameters 1.5 l scale 50 l scalea 50 l scaleb
Vessel diameter (m) 0.129 0.375 0.375
Impeller diameter (m) 0.067 0.150 0.150
Static height of broth (m) 0.115 0.453 0.453
Impeller type Turbine rushton Turbine rushton Turbine rushton
Number of impellers 1 2 2
Geometric factor (fc ) 0.61 0.92 0.92
Reynolds number 2.6 × 104 7.6 × 104a 7.6 × 104b
Rotation speed of impeller (rpm) 350 205a 205b
Aeration rate (l min−1 ) 1.5 20a 50b
Cell dry wt. (g l−1 ) 3.0 ± 1.4 2.0 ± 1.2 4.0 ± 1.3
Expression level of Opc (%) 28 ± 5.4 27 ± 5.1 32 ± 5.1
Production of Opc (mg l−1 ) 418 ± 43 257 ± 27 520 ± 27
Productivity of Opc (mg l−1 h−1 ) 49 ± 4.3 26 ± 3.5 52 ± 3.5
The values for the 1.5 l scale were determined experimentally.
a Calculated parameter for 50 l scale using the P/V scale-up criteria.
b Calculated parameter for 50 l scale using the K a scale-up criteria.
L
 R.E. Pérez et al. / Journal of Biotechnology 127 (2006) 109–114
Fig. 2. SDS-PAGE (12.5%) of samples taken during the 50 l fer-
mentation process using the KL a scale-up criteria. Lane 1: purified
recombinant Opc protein; lane 2: starting culture 0 h; lane 3: sam-
ple taken at 2 h of fermentation process; lane 4: sample taken at 4 h
of fermentation process; lane 5: sample taken at 6 h of fermentation
process; lane 6: sample taken at 8 h of fermentation process; lane 7:
sample taken at 10 h of fermentation process; lane 8: sample taken
at 12 h of fermentation process.
During the first 6 h of growth an increase in the opti-
cal density (cell dry weight) was observed. The max-
imum value of the specific growth rate was obtained
during the first 2 h of growth (μ = 1.73 h−1 ), being
stabilized during the rest of the fermentation process
at μ = 1.07 h−1 . The levels of expression for the Opc
protein production were increased through the fermen-
tation process, reaching the highest productivity level
after 10 h of growth.
At 50 l scale (using the KL a scale-up criteria),
4 g cell dry wt l−1 , 32% level of recombinant Opc
protein expression, 520 mg l−1 volumetric production
and 52 mg l−1 h−1 productivity, were obtained. The
biomass yield on substrate and the product yield on
substrate values were 0.36 g biomass/g substrate and
0.052 g product/g substrate, respectively. The volumet-
ric production and productivity values were similar in
both culture scales; however the biomass yield on sub-
strate and the product yield on substrate increased in 1.3
and 1.2 times, respectively, in the 50 l scale compared
with the 1.5 l culture scale.
These values are comparable with those obtained for
other recombinant proteins produced in E. coli such as
gp-41 (Narciandi and Delgado, 1992; Narciandi et al.,
1993; Narciandi, 1996) and the P64k from N. meningi-
tidis (Espinosa et al., 2002), where the proteins in study
become “toxic” for the cell. It has been reported that in
such cell culture system, higher optical densities (cell
dry weight) have been achieved (Bass and Yansura,
2000).
113
In conclusion, for this highly aerobic fermentation,
scale-up based on keeping a constant overall volumetric
mass transfer coefficient (KL a) was successful whereas
scale-up based on keeping the specific power input con-
stant was not satisfactory. Clearly, the oxygen transfer
had significant influence on production of the target
protein even though other aspects of the fermentation
(e.g. final biomass concentration) were not particularly
affected by oxygen transfer. As a result, we have estab-
lished a fermentation process for the production of the
recombinant Opc protein from N. meningitidis to be
used as an antigen in future vaccine formulations in
humans.
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