Recombinant production of Streptococcus equisimilis streptokinase by Streptomyces lividans.

Abstract

Streptokinase (SK) is a potent plasminogen activator with widespread clinical use as a thrombolytic agent. It is naturally secreted by several strains of beta-haemolytic streptococci. The low yields obtained in SK production, lack of developed gene transfer methodology and the pathogenesis of its natural host have been the principal reasons to search for a recombinant source for this important therapeutic protein. We report here the expression and secretion of SK by the Gram-positive bacterium Streptomyces lividans. The structural gene encoding SK was fused to the Streptomyces venezuelae CBS762.70 subtilisin inhibitor (vsi) signal sequence or to the Streptomyces lividans xylanase C (xlnC) signal sequence. The native Vsi protein is translocated via the Sec pathway while the native XlnC protein uses the twin-arginine translocation (Tat) pathway.
SK yield in the spent culture medium of S. lividans was higher when the Sec-dependent signal peptide mediates the SK translocation. Using a 1.5 L fermentor, the secretory production of the Vsi-SK fusion protein reached up to 15 mg SK/l. SK was partially purified from the culture supernatant by DEAE-Sephacel chromatography. A 44-kDa degradation product co-eluted with the 47-kDa mature SK. The first amino acid residues of the S. lividans-produced SK were identical with those of the expected N-terminal sequence. The Vsi signal peptide was thus correctly cleaved off and the N-terminus of mature Vsi-SK fusion protein released by S. lividans remained intact. This result also implicates that the processing of the recombinant SK secreted by Streptomyces probably occurred at its C-terminal end, as in its native host Streptococcus equisimilis. The specific activity of the partially purified Streptomyces-derived SK was determined at 2661 IU/mg protein.
Heterologous expression of Streptococcus equisimilis ATCC9542 skc-2 in Streptomyces lividans was successfully achieved. SK can be translocated via both the Sec and the Tat pathway in S. lividans, but yield was about 30 times higher when the SK was fused to the Sec-dependent Vsi signal peptide compared to the fusion with the Tat-dependent signal peptide of S. lividans xylanase C. Small-scale fermentation led to a fourfold improvement of secretory SK yield in S. lividans compared to lab-scale conditions. The partially purified SK showed biological activity. Streptomyces lividans was shown to be a valuable host for the production of a world-wide important, biopharmaceutical product in a bio-active form.

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BioMed Central
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Microbial Cell Factories
Open Access
Research
Recombinant production of Streptococcus equisimilis streptokinase
by Streptomyces lividans
Elsa Pimienta1, Julio C Ayala1, Caridad Rodríguez1, Astrid Ramos1, Lieve Van
Mellaert2, Carlos Vallín*1 and Jozef Anné2
Address: 1Laboratorio de Genética, Departamento de Investigaciones Biomédicas, Centro de Química Farmacéutica. Ciudad de la Habana, Cuba
and 2Laboratory of Bacteriology, Rega Institute, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Email: Elsa Pimienta - epimienta@infomed.sld.cu; Julio C Ayala - julio.ayala@cnic.edu.cu; Caridad Rodríguez - caridad.rodriguez@cnic.edu.cu;
Astrid Ramos - astridrm@yahoo.com; Lieve Van Mellaert - lieve.vanmellaert@rega.kuleuven.be; Carlos Vallín* - val@infomed.sld.cu;
Jozef Anné - jozef.anne@rega.kuleuven.be
* Corresponding author
Abstract
Background: Streptokinase (SK) is a potent plasminogen activator with widespread clinical use as a
thrombolytic agent. It is naturally secreted by several strains of beta-haemolytic streptococci. The low
yields obtained in SK production, lack of developed gene transfer methodology and the pathogenesis of its
natural host have been the principal reasons to search for a recombinant source for this important
therapeutic protein. We report here the expression and secretion of SK by the Gram-positive bacterium
Streptomyces lividans. The structural gene encoding SK was fused to the Streptomyces venezuelae CBS762.70
subtilisin inhibitor (vsi) signal sequence or to the Streptomyces lividans xylanase C (xlnC) signal sequence.
The native Vsi protein is translocated via the Sec pathway while the native XlnC protein uses the twin-
arginine translocation (Tat) pathway.
Results: SK yield in the spent culture medium of S. lividans was higher when the Sec-dependent signal
peptide mediates the SK translocation. Using a 1.5 L fermentor, the secretory production of the Vsi-SK
fusion protein reached up to 15 mg SK/l. SK was partially purified from the culture supernatant by DEAE-
Sephacel chromatography. A 44-kDa degradation product co-eluted with the 47-kDa mature SK. The first
amino acid residues of the S. lividans-produced SK were identical with those of the expected N-terminal
sequence. The Vsi signal peptide was thus correctly cleaved off and the N-terminus of mature Vsi-SK fusion
protein released by S. lividans remained intact. This result also implicates that the processing of the
recombinant SK secreted by Streptomyces probably occurred at its C-terminal end, as in its native host
Streptococcus equisimilis. The specific activity of the partially purified Streptomyces-derived SK was
determined at 2661 IU/mg protein.
Conclusion: Heterologous expression of Streptococcus equisimilis ATCC9542 skc-2 in Streptomyces lividans
was successfully achieved. SK can be translocated via both the Sec and the Tat pathway in S. lividans, but
yield was about 30 times higher when the SK was fused to the Sec-dependent Vsi signal peptide compared
to the fusion with the Tat-dependent signal peptide of S. lividans xylanase C. Small-scale fermentation led
to a fourfold improvement of secretory SK yield in S. lividans compared to lab-scale conditions. The
partially purified SK showed biological activity. Streptomyces lividans was shown to be a valuable host for
the production of a world-wide important, biopharmaceutical product in a bio-active form.
Published: 5 July 2007
Microbial Cell Factories 2007, 6:20doi:10.1186/1475-2859-6-20
Received: 12 April 2007
Accepted: 5 July 2007
This article is available from: http://www.microbialcellfactories.com/content/6/1/20
© 2007 Pimienta et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Background
Streptokinases are proteins translocated to the growth
medium by many strains of beta-haemolytic streptococci.
Streptokinase is not an enzyme per se but rather a potent
activator that interacts with plasminogen to form a stoi-
chiometric 1:1 complex. This interaction results in the
activation of plasminogen to plasmin, which is the active
fibrinolytic component of the circulatory system [1]. SK
was the first drug introduced as a therapy for acute myo-
cardial infarction more than 40 years ago [2]. It is now the
leading fibrinolytic agent in the treatment of thromboem-
bolic conditions [3] and is included in the World Health
Organization Model List of Essential Medicines.
The Streptococcus equisimilis H46A skc gene encoding strep-
tokinase has been cloned and expressed in several heterol-
ogous hosts due to the pathogenicity of its natural host.
Haemolytic streptococci secrete several toxins that com-
plicate the downstream purification. Besides, genetic
modification of the natural host is restricted as rather few
genetic tools are available. As a result, the recombinant
production of this protein in E. coli has been widely used,
including the use of the protein SKC-2 naturally secreted
by Streptococcus equisimilis ATCC 9542 [4,5]. High-level
expression of skc in E. coli has been reported, but the for-
mation of inclusion bodies consisting of highly aggre-
gated SK molecules makes its recovery in an active form
difficult [4,5]. High level of intracellular SK has also been
obtained during continuous fermentation of recombinant
Pichia pastoris but protein recovery requires cell lysis [6].
Since the recovery of extracellular proteins is generally eas-
ier than that of cytoplasmic proteins, the expression and
subsequent secretion of SK have been studied in several
heterologous hosts like Escherichia coli, Bacillus subtilis and
Pichia pastoris [7-9]. In case of B. subtilis, the use of the six-
extracellular-protease-deficient strain, WB600, greatly
improved the yield of recombinant SK. The protein was
also secreted into the culture medium by P. pastoris, but it
was found to be heavily glycosylated. The biological activ-
ity of both secreted streptokinases was proved. A recent
study using Schizosaccharomyces pombe as host, reported
the expression of SK and its secretion into the periplasmic
fraction without glycosylation and significant degradation
or modification. However, conventional chromato-
graphic approaches used before to purify SK from other
hosts were inadequate because of cofractionation of a few
proteins of similar size with SK through all the chromato-
graphic steps [10].
As it is not possible to predict which host will be the best
for the production of a protein, the aim of this work was
to evaluate Streptomyces lividans as host for recombinant
production of SK. S. lividans has been successfully used for
the production of several proteins of bacterial and eukary-
otic origin [11-13]. The advantages of the S. lividans host
include its natural ability to secrete high levels of bioactive
molecules into the extracellular medium, limited protease
activity, its biological safety and well-established fermen-
tation technology [14]. In the present study, the S. lividans
system has been tested for the secretory production of the
streptokinase from Streptococcus equisimilis group C by
using the Sec and the recently described twin-arginine
translocation (Tat) pathway in S. lividans [15]. The
sequence encoding mature SK was fused to the Sec-
dependent signal sequence of Streptomyces venezuelae
CBS762.70 subtilisin inhibitor [16] and the twin-arginine
signal sequence of S. lividans xylanase C [17], respectively.
SK production in S. lividans was evaluated and purifica-
tion of the secreted SK protein was carried out.
Results
Construction of SK expression/secretion vectors
In order to establish the expression and secretion of SK
from Streptococcus equisimilis ATCC9542 in S. lividans, the
skc-2 gene was amplified by PCR using chromosomal
DNA as template and finally cloned in appropriate vectors
in Streptomyces. The constructed expression/secretion vec-
tors pOVsiSK and pOXlnCSK encode the fusion proteins
Vsi-SK and XlnC-SK, respectively (Table 1). Vsi-SK consists
of the Sec-dependent Vsi signal peptide, the first two
amino acid residues of mature Vsi followed by the mature
SK, while XlnC-SK is composed of the Tat-dependent
XlnC signal peptide, the first three amino acids of mature
XlnC and mature SK. In this way, the signal peptidase
cleavage sites of Vsi and XlnC are remained and a proper
processing of the precursor proteins is achieved. Both
fusion genes were placed under control of the vsi pro-
moter, of which has been proved that it efficiently pro-
motes heterologous gene expression [13,16].
Secretion of SK by S. lividans
S. lividans transformants carrying pOVsiSK and pOXl-
nCSK, respectively, were grown at lab scale in rich BTSB
medium and at several time intervals the presence of SK in
the culture filtrate was assessed by Western blot analysis.
A clear SK-specific band of about 47 kDa and smaller
immunoreactive bands of approximately 44 kDa and 32
kDa could be observed in culture supernatant of S. lividans
[pOVsiSK] at 30 and 40 h of growth (Fig. 1, lanes 3 and
4). SK was faintly detectable in culture filtrate of S. lividans
[pOXlnCSK] upon 30 and 40 h growth (Fig. 1, lanes 5 and
6). No SK-specific immunoreactive proteins could be
detected in cell lysates of S. lividans carrying pOVsiSK or
pOXlnCSK (data not shown), which indicated that the
produced preproteins did not accumulate inside the cell
and were efficiently translocated through the cell mem-
brane.

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The amount of SK secreted by the S. lividans transform-
ants, measured by means of ELISA, reached a maximum
around 40 h and then decreased. Upon 40 h growth, up to
4 mg SK/l medium was measured in culture supernatant
of S. lividans [pOVsiSK] (Fig. 2), while 100–150 µg SK/l
was detected in the extracellular fraction of 40-h S. lividans
[pOXlnCSK] cultures. These results indicate that SK can be
translocated via both the Sec and the Tat pathway in S. livi-
dans, but the Sec-routed secretion leads to higher levels of
the recombinant protein.
In consequence of the poor SK yield using the XlnC signal
peptide as mediator for translocation, only S. lividans
[pOVsiSK] was tested under fermentation conditions.
Using small-scale fermentation conditions, the secretory
SK production reached up to 15 mg SK/l, which corre-
sponds to a fourfold improvement of secretory SK yield in
S. lividans compared to lab-scale conditions.
Secretory yield of recombinant SK correlated with biomass of Streptomyces lividans [pOVsiSK] grown in lab-scale condi-tions
Figure 2
Secretory yield of recombinant SK correlated with
biomass of Streptomyces lividans [pOVsiSK] grown in
lab-scale conditions. SK concentration was determined by
means of ELISA. Growth was estimated by measuring bio-
mass dry weight (mg/ml), standard errors were between 0.1
and 0.2.
Immunodetection of SK in extracellular fractions
Figure 1
Immunodetection of SK in extracellular fractions.
Proteins from culture supernatants were precipitation with a
mixture of chloroform and methanol (1:3, v/v). In each lane,
proteins according to 100 µl spent culture medium were
loaded. Lane 1, S. lividans [pOW15] 40 h; lane 2, 60 ng SK
standard; lane 3, S. lividans [pOVsiSK] 30 h; lane 4, S. lividans
[pOVsiSK] 40 h; lane 5, S. lividans [pOXlnCSK] 30 h; lane 6, S.
lividans [pOXlnCSK] 40 h.
Table 1: Plasmids used in this study.
NameRelevant propertiesSource or reference
pGEM-SK
pBS-CBSS
pGEM®-T Easy derivative containing the Streptococcus equisimilis skc-2 gene
pBluescript KS(+) derivative containing the Streptomyces venezuelae vsi promoter and part of the mature
vsi gene
pBluescript KS(+) derivative containing the Streptomcyes venezuelae vsi promoter and the signal
sequence of Streptomyces lividans xlnC
pBSVX derivative containing a unique EcoRI site downstream of the signal sequence of Streptomyces
lividans xlnC
Escherichia coli-Streptomyces shuttle vector, multiple cloning site, ApR, TsrR
pUWL-218 derivative E. coli-Streptomyces shuttle vector containing the oriT fragment for interspecies
DNA conjugation.
pOW15 derivative containing the Streptomyces venezuelae vsi promoter, and signal sequence, and the
Streptococcus equisimilis skc-2 gene
pOW15 derivative containing the Streptomyces venezuelae vsi promoter, the Streptomyces lividans xlnC
signal sequence and the Streptococcus equisimilis skc-2 gene
This work
[16]
pBSVX [17]
pBSVXM This work
pUWL-218
pOW15
[40]
Rosabal et al., unpublished.
pOVsiSKThis work
pOXlnCSKThis work

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Purification of recombinant SK secreted by S. lividans
Having defined the fermentation conditions for the secre-
tory production of recombinant SK, the protein was puri-
fied from the extracellular culture fraction. The protein
fraction obtained through ammonium sulfate precipita-
tion (45% saturation) was dissolved in 20 mM Tris-HCl
(pH 6.0), dialyzed against the same buffer and then
applied on a DEAE-Sephacel column. The proteins were
eluted in 20 mM Tris-HCl, 150 mM NaCl, pH 6.0. Sam-
ples from the various purification steps were analysed by
SDS-PAGE followed by Coomassie staining (Fig. 3A) and
immunodetection of the recombinant SK using a mono-
clonal anti-SK antibody (Fig. 3B). Samples from culture
supernatant and 45% ammonium sulphate saturation
fraction of S. lividans TK 24 [pOW15] were included as
negative controls (Fig. 3, lanes 1 and 2). This experiment
revealed that the purified proteins correspond to SK.
Pooled elution fractions with a purity grade of 58% con-
tained the 44-kDa degradation product which co-eluted
with the 47-kDa mature SK.
In order to determine the specificity of signal peptidase
processing and the nature of the approximately 44-kDa
protein, an N-terminal sequence was carried out on both
the 47- and 44-kDa proteins obtained from S. lividans TK
24 [pOVsiSK] culture supernatant. N-terminal residues of
both purified proteins were those predicted from the
sequence (Table 2). The Vsi signal peptide was thus cor-
rectly cleaved off and the N-terminus of mature Vsi-SK
released by S. lividans remained intact. This result also
implicates that the processing of mature Vsi-SK secreted
by Streptomyces occurred at its C-terminal end.
The specific activity of the partially purified proteins
secreted by S. lividans [pOVsiSK] was amounted to 2661
IU/mg protein (Table 3). In consequence of incompatibil-
ity between the crude culture medium and the chromoge-
nic substrate assay, we were not able to determine the
initial specific activity of SK.
Discussion
In the present study, it was shown that SK from Streptococ-
cus equisimilis ATCC9542 could be efficiently secreted in a
bio-active form via the Sec pathway in Streptomyces livi-
dans. Sec-routed secretion was obtained by using the regu-
latory signal sequences of S. venezuelae CBS762.70
subtilisin inhibitor gene. The Tat translocation route was
also tested for the secretion of SK in S. lividans by means
of a fusion of SK to the Tat-dependent signal peptide of S.
lividans xylanase C. Yield was about 30 times higher when
the SK was fused to the Sec-dependent Vsi signal peptide
compared to the fusion with the Tat-dependent XlnC sig-
nal peptide. Although the use of the Tat pathway in most
cases does not result in higher production yield compared
to Sec-mediated secretion (e.g. Schaerlaekens et al. 2004),
some proteins need to be secreted via the Tat pathway to
obtain their bio-active conformation. This is the case for
the homologous protein xylanase C [18], but also for the
heterologous enhanced green fluorescent protein (EGFP)
[19].
The maximum level of SK secreted by S. lividans was 15
mg/l of culture, but as a result of incompatibility between
the crude culture medium and the chromogenic substrate
assay, we were not able to determine the initial activity of
Table 2: Amino acid sequence of the fusion region of preVsi-SK
and the N-terminal amino acid sequence of the 47- and 44-kDa
proteins obtained from S. lividans TK 24 [pOVsiSK] culture
supernatant.
ProteinAmino acid sequence
PreVsi-SK (fusion region)
N-terminus 47-kDa rSK
N-terminus 44-kDa rSK
...A Q A ↓ E A I A G P E W L L...
E A I A G P E W L L...
E A I A G P E W L L...
↓: Signal peptidase cleavage site, SK sequence in italics
Purification of extracellular SK from S. lividans culture super-natants upon small-scale fermentation
Figure 3
Purification of extracellular SK from S. lividans cul-
ture supernatants upon small-scale fermentation. (A)
10% SDS-PAGE stained with Coomassie blue R-250, and (B)
Immunoblotting analysis using a monoclonal anti-SK antibody.
Lane 1, 25 µg of crude extract of S. lividans TK24 [pOW15];
lane 2, 25 µg of material precipitated with (NH4)2SO4 of S.
lividans TK24 [pOW15]; lane 3, 25 µg of crude extract of S.
lividans TK24 [pOVsiSK]; lane 4, 25 µg of proteins precipi-
tated with (NH4)2SO4 of S. lividans TK24 [pOVsiSK]; lane 5,
25 µg of pooled anion exchange chromatography protein
fractions with 58% purity; lane 6, 1 µg of SK standard; lane 7,
Broad-range protein molecular weight markers.
B
A

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Streptomyces-derived SK. SK secreted by recombinant S.
lividans was partially purified (58% purity) and was found
biologically active with an specific activity of 2661 IU/mg
protein. In addition, it is not possible to compare reliably
the plasminogen activity of the partially purified SK
secreted by Streptomyces with the initial SK activity secreted
by other hosts like: Streptococcus equisimilis (100–150 IU/
ml), E. coli (1000–1500 IU/ml), P. pastoris (3200 IU/m1)
or S. pombe (2450 IU/m1) [9,10]. However, it is possible
to establish a relative comparison with the total yield
(24.5 mg/l) and the initial specific activity of SK secreted
by S. pombe (1581 IU/mg protein) [10].
SK has a tendency to degrade very easily [20,21]. Several
hosts, including the native host Streptococcus equisimilis,
produce at least two major forms of SK [7,8,22]: the intact
mature SK with a molecular mass of 47 kDa and a 44-kDa
degradation product. This degradation product lacks 31 or
32 C-terminal residues whereas it retains the plasminogen
activation capability [23]. Furthermore, C-terminal dele-
tion mutants of SK lacking 40 [24] or 41 amino acids [25]
exhibited normal plasminogen activator function. In
addition to the 47- and 44-kDa bands, a 32-kDa degrada-
tion product was detected by Western blot. Since SK pro-
teins which lack 18 or more N-terminal or 51 or more C-
terminal amino acid residues are unlikely to be effective
thrombolytic agents [24], the 32-kDa SK-related protein
missing about 135 aa residues was not further investi-
gated.
It was demonstrated that the post-translational modifica-
tion at the C-terminus of native SK was caused by chymo-
trypsin-like activity [23]. Similar degradation of
recombinant SK has been also reported to occur in heter-
ologous hosts such as Streptococcus sanguis [23] and E. coli
[26]. Chymotrypsin-like activity and several genes encod-
ing chymotrypsin-like serine proteases have been reported
in S. lividans 66 [27,28]. Since the first amino acid residues
of the S. lividans-produced Vsi-SK were identical to those
of the expected N-terminal sequence, the recombinant
protein was proteolytically degraded at its C-terminal end.
In case of recombinant SK production in Lactococcus lactis,
the protease susceptibility and hence the productivity of
SK was dependent on the pH of the culture and the initial
phosphate concentration of the medium. Suppression of
the acid tolerance response, by which protease expression
is induced, enhanced the SK yield 2.5 fold [29]. Results of
a differential scanning calorimetry study on E. coli-derived
recombinant S. equisimilis SK firmly indicated that at neu-
tral and basic pH, the recombinant SK from Streptococcus
equisimilis group C (ATCC 9542) has four domains,
whereas gentle changes in the experimental conditions,
such as mild acidification or increase in the NaCl concen-
tration, decreased this number [30]. Consequently, pH
and ionic strength of the production medium define the
conformational status of SK and are thus important fac-
tors determining the protease susceptibility of the recom-
binant protein.
The specific activity of the partially purified SK (58%
purity) secreted by S. lividans [pOVsiSK] was determined
at 2661 IU/mg protein. We believe that further up-scaling
of the fermentation process and optimisation of produc-
tion medium and purification protocol, will surely
improve yield of recombinant bio-active SK in S. lividans.
Conclusion
Heterologous expression of Streptococcus equisimilis
ATCC9542 skc-2 in Streptomyces lividans was successfully
achieved. SK can be translocated via both the Sec and the
Tat pathway in S. lividans, but yield was about 30 times
higher when the SK was fused to the Sec-dependent Vsi
signal peptide compared to the fusion with the Tat-
dependent signal peptide of S. lividans xylanase C. Small-
scale fermentation led to a fourfold improvement of secre-
tory SK yield in S. lividans compared to lab-scale condi-
tions. The plasminogen activity of the partially purified SK
(58% purity) secreted by S. lividans [pOVsiSK] was deter-
mined at 2661 IU/mg protein. Once more, Streptomyces
lividans was shown to be a valuable host for the produc-
tion of a world-wide important, biopharmaceutical prod-
uct in a bio-active form.
Methods
Bacterial strains and growth conditions
E. coli TG1 was used as host for cloning purposes. Culture
conditions for E. coli were as described by Sambrook et al.
[31]. Streptococcus equisimilis ATCC9542 cells were grown
as described by Estrada et al. [4]. Streptomyces lividans
TK24 was selected as host for heterologous protein pro-
duction. Protoplast formation and subsequent transfor-
Table 3: Secretory production of recombinant SK by S. lividans TK 24 [pOVsiSK].
Sample Volume (ml)SK activity (IU/ml)Protein concentration
(mg/ml)
ELISA SK (mg/ml) Specific activity
(IU/mg)
Culture supernatant
DEAE eluates with 58% purity
1000
16
ND1.04
0.17
0.015
0.021
ND
444 ± 242661 ± 29
ND: No determined.

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mation of S. lividans were carried out as described by
Kieser et al. [32]. Regeneration of S. lividans protoplasts
and selection of transformants was carried out on MRYE
medium [33]. When appropriate, thiostrepton (50 µg/ml
in solid medium or 10 µg/ml in liquid medium) was
added. Spore suspensions of S. lividans TK24 and deriva-
tives were stored at -70°C in 20% (v/v) glycerol. Primary
cultures of S. lividans strains were routinely cultured for 72
h (28°C, 240 rpm) in BTSB, which is a modified version
of the medium described by Dyson and Schrempf [34]:
10% sucrose, 1% yeast extract, 1% glucose, 0.5% NaCl,
0.5% soya flour, 1.7% tryptone, 0.25% K2HPO4, pH 7.2.
For monitoring recombinant protein expression and
secretion, 1-ml primary cultures were inoculated to 0.5-L
shake flask containing 0.1 L of BTSB medium and grown
for 40 h at 28°C and 300 rpm. For production of SK, the
recombinant strain was cultured for 48 h (28°C, 350
rpm) in a 2.5-L MBR reactor containing 1.5 L BTSB
medium. The pH was controlled at 7.0 by the addition of
5 N NaOH.
Plasmid construction and recombinant DNA technology
DNA manipulations were carried out following standard
procedures [31,32]. Restriction endonucleases and DNA-
modifying enzymes were from Invitrogen and Roche
Diagnostics. Upon isolation of S. equisimilis ATCC9542
chromosomal DNA as described by Estrada et al. [4], the
skc-2 gene was PCR-amplified from the chromosome
under standard conditions using the oligonucleotides SK-
F1 and SK-R1 (see Table 4) and Taq polymerase. To allow
in-frame fusion of the skc-2 gene downstream the signal
sequences, the oligonucleotide SK-F1 was designed with
an EcoRV site (GATATC) in its 5'end. Consequently, a
silent mutation was introduced in the first codon of the
mature part of the skc-2 gene: ATT was replaced by ATC,
both encoding isoleucine.
The 1245-bp PCR fragment was ligated into pGEM®-T
Easy (Promega) and the resulting plasmid was denomi-
nated pGEM-SK. The DNA sequence was verified using the
Thermo Sequenase Primer Cycle Sequencing Kit with 7-
deaza-dGTP on an ALFexpress apparatus (Amersham Bio-
sciences, Rainham, UK). Subsequently, the 1.3-kb EcoRV/
EcoRI fragment of pGEM-SK was cloned into pBS-CBSS
[16] successively treated with DraII, Klenow polymerase
and EcoRI. The unique DraII site in pBS-CBSS is located
two codons downstream the signal peptidase recognition
site. skc-2 was also cloned into pBSVXM, a derivative of
plasmid pBSVX [17] missing an EcoRI site. To remove the
EcoRI site located upstream the vsi promoter in pBSVX, a
site-directed mutagenesis was carried out by means of
PCR using Pfu polymerase and the mutagenic oligonucle-
otides PBSXylE-F and -R (Table 4), which contain the
desired mutation. As such, a unique EcoRI site located
downstream the S. lividans xlnC signal sequence was avail-
able. In order to insert the skc-2 gene fused to the third
codon of mature xlnC, the vector pBSVXM was digested
with NsiI, treated with T4 DNA polymerase removing the
3'-protruding ends and finally treated with EcoRI. DNA
sequence analyses of the newly constructed fusion genes
confirmed their correctness.
Finally, both expression/secretion cassettes were isolated
as BamHI/EcoRI-fragments and ligated in BamHI/EcoRI-
digested pOW15. The vector with the vsi signal sequence
was designated pOVsiSK and the vector with the xlnC sig-
nal sequence was denominated pOXlnCSK. Plasmids
used in this study are listed in Table 1.
Detection of SK
The detection of SK in culture supernatants and cell lysates
of S. lividans transformed with pOVsiSK or pOXlnCSK was
performed using Western Blot and immunodetection. Gel
electrophoresis of proteins was carried out on 10% SDS-
polyacrylamide gels [35]. Separated proteins were visual-
ized by Coomassie brilliant blue staining or transferred to
a Hybond™-C extra membrane (GE Healthcare) by using
a semidry transfer cell (Biometra) according to the manu-
facturer's recommendations. SK was detected using a
mouse anti-SK monoclonal antibody (produced by
Center for Genetic Engineering and Biotechnology, Sancti
Spiritus, Cuba). HRP-conjugated goat anti-mouse anti-
body (Promega) was used as secondary antibody. Immu-
noreactive bands were visualized by brief exposure to 3,3-
diaminobenzidine or 4-chloro naftol (Sigma). Cell lysates
were obtained according to Pimienta et al. [36]. The pro-
tein content of culture supernatants, cell lysates and puri-
fied fractions was determined using the Bradford method
[37].
Table 4: Oligonucleotides used in this study.
Name Sequence (5'-3' direction)Restriction sites (in italic)
SK-F1
SK-R1
PBSXylE-F
PBSXylE-R
GATATCGCTGGACCTGAGTGGCTG
AGATCTTTATTTGTCGTTAGGGTTATCAG
CCGGGCTGCAGGAAGTCGATTCGGGAGCG
CGCTCCCGAATCGACTTCCTGCAGCCCGG
EcoRV
BglII
-
-
The mutagenic base is in bold face.

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Page 7 of 8
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The molecular size and the purification degree of recom-
binant SK protein were estimated from densitometric
scanning of Coomassie brilliant blue-stained gels using a
GENE GENIUS gel documentation system and GeneTools
software (Syngene).
SK present in culture supernatants or anion exchange
chromatography eluates was quantified by means of a
general sandwich ELISA protocol (Abrahantes et al.
unpublished results). The SK standard was kindly sup-
plied by the Development Division, Center for Genetic
Engineering and Biotechnology, Havana, Cuba. The coef-
ficient of variation of the ELISA tests was less than 10%.
SK activity was monitored spectrophotometrically at 405
nm in a coupled SK-plasminogen assay employing the
chromogenic substrate S-2251 (Kabi, Sweden) according
to Hernández et al. [38]. The specific activity (IU/mg) was
calculated by dividing the SK activity (IU/ml) with protein
concentration (mg/ml).
Protein purification and chromatography
For purification of SK from recombinant S. lividans cul-
tures, strains were grown for 2 days in 200 ml BTSB
medium. Then, cultures were centrifuged and the myc-
elium was resuspended in 0.1 L of water. This suspension
was transferred to 1.5 L BTSB in the MBR reactor and bac-
terial growth was continued for 2 days. Culture superna-
tant proteins were precipitated by addition of (NH4)2SO4
(45% saturation, 4°C) and collected by centrifugation
(Hettich Universal 32R centrifuge, Sorvall, 1620A rotor,
4°C, 20 min, 8000 × g). The protein pellets were left over-
night at 4°C in 0.1 L of 20 mM Tris-HCl buffer, pH 6.0.
Then, the protein solution was dialyzed against 20 mM
Tris-HCl buffer (pH 6.0) at 4°C for 20 h and was finally
applied on a DEAE Sephacel column equilibrated with 20
mM Tris-HCl buffer, pH 6.0. The column was extensively
washed with the mentioned buffer followed by 1 column
volume of 20 mM Tris-HCl, 20 mM NaCl, pH 6.0. SK pro-
tein elution from the DEAE Sephacel column was carried
out with 3 column volumes of 20 mM Tris-HCl, 150 mM
NaCl, pH 6.0 at a flow rate of 0.5 ml/min. One ml frac-
tions were collected. Fractions containing SK with a simi-
lar degree of purity, determined by means of SDS-PAGE
followed by Coomassie staining, were pooled.
N-terminal amino acid sequence analysis
The purified SK was subjected to SDS-PAGE and blotted
on a Hybond-P membrane (GE healthcare) as described
by Ausubel et al. [39]. After Coomassie staining, the rele-
vant protein bands were excised and subjected to sequenc-
ing. The N-terminal amino acid sequence of recombinant
SK was determined by Edman degradation using an auto-
matic 477A-1201 protein sequencing system (Applied
Biosystems).
Competing interests
The author(s) declare that they have no competing inter-
ests.
Authors' contributions
EP participated in the design of the study, performed clon-
ings, strain constructions and fermentation, analyzed the
data and wrote the manuscript. JCA participated in design
and performance of protein purification. CR participated
in design and performance of protein purification. AR par-
ticipated in design and performance of growth conditions.
LVM, CV and JA participated in design of the study and
commented the manuscript. All authors have read and
approved the manuscript.
Acknowledgements
Part of this work was supported by the Research Project ZEIN2002PR262
of the "Vlaams interuniversitaire raad"/University Development Coopera-
tion in collaboration" with the Rega Institute, Katholieke Universiteit Leu-
ven, Belgium. We thank Prof. Paul Proost (Rega Institute, K.U. Leuven) for
N-terminal amino acid determinations. The assistance provided by Ing.
Lázara Muñoz, Bsc. Dinorah Torres, Ing. Jorge Valdés and Dr. Eduardo Mar-
tínez from the Development Division, Center for Genetic Engineering and
Biotechnology, Cuba is thankfully acknowledged.
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