Yeast
Yeast 2004; 21: 1343–1358.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/yea.1184
Research Article
Expression and secretion of a prokaryotic protein
streptokinase without glycosylation and degradation in
Schizosaccharomyces pombe
Raj Kumar and Jagmohan Singh*
Institute of Microbial Technology, Sector 39A, Chandigarh 160036, India
*Correspondence to:
Jagmohan Singh, Institute of
Microbial Technology, Sector
39A, Chandigarh 160036, India.
E-mail: [email protected]
Received: 15 June 2004
Accepted: 24 September 2004
Abstract
Streptokinase (SK) is an important thrombolytic protein that is secreted by pathogenic
strains of Streptococcus. Expression of streptokinase has been so far attempted in
Pichia pastoris, Escherichia coli and Bacillus subtilis and shown to yield protein that
was either highly glycosylated or degraded. Since the fission yeast, Schizosaccharomyces pombe, shares several molecular characteristics with higher eukaryotes, we
decided to express the streptokinase gene in this yeast. A chimeric gene comprising the signal sequence of the Plus pheromone of Sz. pombe fused in-frame with
the mature streptokinase from Streptococcus sp. was constructed and inserted into
the expression vector containing the thiamine-regulated promoter. We obtained a
high level of expression of streptokinase comparable to that in E. coli and P. pastoris, with 50–100% processing of the signal sequence and secretion of the mature
streptokinase into the periplasmic fraction. The mature enzyme co-migrates with
the authentic mature SK in SDS gels, lacks any major modification and is functional. Importantly, a higher level of expression under stationary phase conditions and
improved extractability of the mature and undegraded streptokinase was achieved in
a novel mutant of Sz. pombe defective for a potent extracellular protease activity. We
suggest that the unique vector/strain system developed here could be advantageous
for large-scale production of prokaryotic proteins without significant modification or
degradation in Sz. pombe. Copyright  2004 John Wiley & Sons, Ltd.
Keywords: streptokinase; expression; secretion; P-factor;
Schizosaccharomyces pombe
Introduction
Expression of heterologous genes in bacteria and
yeast is a powerful tool, not only for producing proteins of therapeutic and commercial interest but also as drug discovery and molecular
tools for biological research. Among bacteria,
Escherichia coli and Bacillus subtilis (Shatzman,
1993) have been exploited extensively because
of short generation time and inexpensive growth
media. However, bacterial expression systems pose
problems such as endotoxin production, frequent
suboptimal codon usage, formation of inclusion
bodies and lack of post-translational modifications, which are generally critical for the structure and function of eukaryotic proteins. These
Copyright  2004 John Wiley & Sons, Ltd.
problems have shifted the focus to lower eukaryotes such as yeasts. The budding yeast Saccharomyces cerevisiae has been used extensively
(Shatzman, 1993). However, the levels of expression achieved are generally low and recombinant
proteins are frequently hyperglycosylated (Shatzman, 1993). Likewise, the methanol-inducible promoters of the methylotropic yeasts Pichia pastoris
and Hansenula polymorpha offer the advantage
of inexpensive growth media (Shatzman, 1993).
Moreover, these yeasts can achieve high cell density and multiple integrations into the genome,
allowing higher yields of recombinant proteins.
However, expression is frequently accompanied by
non-physiological post-translational modifications
(Shatzman, 1993).
1344
In this regard, the fission yeast Sz. pombe
has not been exploited so extensively (Shatzman,
1993; Giga-Hama, 1997a). Sz. pombe is a genetically tractable organism and, in contrast with S.
cerevisiae, it shows several molecular similarities to higher eukaryotes (Russell, 1989; GigaHama, 1997a), e.g. mRNA splicing, similar splice
signals, complex replication origins, centromere
structure, etc. Even genes from humans containing introns can be expressed in Sz. pombe (Lee
and Nurse, 1987). Some examples of expression
of heterologous genes in fission yeast include
human antithrombin III (Broker et al., 1987),
human lipocortin I (Giga-Hama et al., 1994) and
human papillomavirus E7 protein (Tommasino
et al., 1990). However, only a few regulatable
promoters are known in Sz. pombe, the most
widely used being the thiamine-regulated promoter
nmt1 (Forsburg, 1986; Maundrell, 1990, 1993).
Only recently has the homologous pheromonebased secretory signal been exploited in Sz. pombe
(Giga-Hama, 1997b).
Streptokinase (SK) is a potent plasminogen activator, secreted by pathogenic strains of Streptococcus as a 47 kDa protein. The gene skc encoding SK
has been cloned and expressed in several heterologous species (Malke and Ferretti, 1984; Hagenson et al., 1989; Pratap et al., 1996, 2000). Secretion of SK into the culture medium was shown to
occur in E. coli (Malke and Ferretti, 1984; Pratap
et al., 1996). However, post-translational processing close to the C-terminus yielded an additional
protein band of 44 kDa (Malke and Ferretti, 1984;
Pratap et al., 1998). Likewise, when expressed in
P. pastoris, SK was found to be secreted but
also heavily glycosylated (Pratap et al., 2000).
Although glycosylation seemed to enhance the stability of the SK in vitro (Pratap et al., 2000),
the high glycosylation levels may elicit immune
response upon injection.
In this study, we have constructed a chimeric
gene with the N-terminal tag corresponding to
the signal sequence of Plus (P) pheromone of Sz.
pombe (Imai and Yamamoto, 1994), fused in-frame
with the mature streptokinase (Malke and Ferretti,
1984) and expressed it under the control of nmt1
promoter. We have obtained a level of expression comparable to that achieved in E. coli and
P. pastoris, with 50–100% processing of the signal sequence and secretion of the active, mature
Copyright  2004 John Wiley & Sons, Ltd.
R. Kumar and J. Singh
streptokinase into the periplasmic fraction. Significantly, the SK expressed in Sz. pombe exhibits
no modifications and undergoes very little degradation — desirable characteristics for studying structure–function relationships and for commercial
considerations. Most importantly, we have isolated
a mutant defective in a potent extracellular protease
activity in Sz. pombe. Interestingly, this mutant
allows expression of SK under stationary phase
conditions with enhanced solubility, processing and
secretion of the mature SK into the periplasmic
fraction, with no detectable proteolytic degradation.
Materials and methods
Construction of the expression vector
The construction of the chimeric gene containing
the signal sequence of Plus (P) factor of Sz. pombe
and mature streptokinase from Streptococcus equisimilis (Figure 1A) has been described previously
(Mehta and Singh, 1999). XhoI and BamHI sites
were introduced into the primers used for construction of the chimeric gene. The PCR product
was cleaved with XhoI plus BamHI and cloned
into the corresponding sites of pREP3, which
contains the nmt1 promoter (Maundrell, 1993),
yielding the plasmid pJRK1 (Figure 1B). In-frame
fusion and authenticity of PSK gene was confirmed
by nucleotide sequence analysis of the recombinant construct. The resulting recombinant plasmid
pJRK1 was transformed into Sz. pombe and transformants were screened for streptokinase activity
by the skim milk plate assay (Malke and Ferretti,
1984; Pratap and Dikshit, 1998).
Growth conditions for induction of expression
In all experiments the Sz. pombe strain SP837 was
used (genotype: h 90 , leu1-32, ura4D18, ade6-216).
A transformant of this strain harbouring the recombinant plasmid pJRK1 (it contains the S. cerevisiae
LEU2 gene, which complements the leu1 − mutation of Sz. pombe) was inoculated into 20 ml YEA
medium (yeast extract medium containing adenine)
according to Moreno et al. (1991) and incubated at
30 ◦ C for up to 18 h. Overnight culture was further inoculated into fresh 20 ml YEA medium and
grown to OD600 = 0.1. The culture was incubated
for an additional 4–5 hours at 30 ◦ C to reach midlog phase (OD600 = 0.5). In another experiment,
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Expression of streptokinase in Sz. pombe
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Figure 1. (A) Construction and design of the vector expressing the chimera between P factor signal sequence and mature
SK and (B) restriction map of recombinant plasmid pJRK1 containing PSK chimeric gene. (A) The chimera involves fusion
between residues 1–32 of P factor of Sz. pombe (corresponding to its signal sequence) and the mature SK. (B) The chimeric
PCR construct of Plus pheromone and authentic SK (Malke and Ferretti, 1984) was cloned into the XhoI and BamHI sites
of the vector pREP3 to generate the plasmid pJRK1
OD600 at the time of induction was brought up to
0.2, 0.3, 0.4, 0.5, 0.7, 0.9, 1.2 or 1.5. Cells were
harvested by centrifugation and washed three times
with PMA leu− (pombe minimal medium containing adenine but lacking leucine; Moreno et al.
1991). The cells were then resuspended in 20 ml
PMA leu− containing 25 mM thiamine (for gene
repression) or without thiamine (for gene induction) and grown at 30 ◦ C for 15 h.
Preparation of cell extracts
Cells were harvested, washed once with chilled
water and then with buffer A (0.02 M Hepes,
pH 7.5, 0.1 M sodium chloride, 0.002 M EDTA,
0.625% glycerol and 1 mM β-mercaptoethanol).
The cell pellet was then resuspended in 1 ml buffer
A containing protease inhibitors (1 mM PMSF,
1 µg/ml aprotinin, 1 µM pepstatin A and 1 µM
leupeptin). Acid-washed glass beads (equal weight
Copyright  2004 John Wiley & Sons, Ltd.
of pellet, 425–600 mm in diameter, Sigma) were
added and the cells were broken by vortexing at
4 ◦ C after ultracentrifugation at 100 000 × g for
30 min in a TL100.2 rotor of the TL100 table-top
ultracentrifuge. The supernatant (cell extract) was
collected and saved.
Sub-cellular fractionation was carried out according to Moreno et al. (1985). Cells were suspended
in SCE buffer (1 M sorbitol, 0.1 M sodium citrate,
60 mM EDTA and 1% β-mercaptoethanol) containing 0.05 mg/ml Zymolyase-100T (Seikagaku) and
protease inhibitors. Incubation was carried out at
37 ◦ C for 1 h to obtain up to 90% spheroplasting,
followed by centrifugation at 1000 × g for 5 min
at 4 ◦ C. The supernatant, which corresponds to the
solubilized fraction obtained during preparation of
protoplasts according to Moreno et al. (1985), represented the periplasmic protein (PP) fraction. The
pellet was washed with cold buffer A and resuspended in 1 ml of buffer A containing protease
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1346
inhibitors. Acid-washed glass beads were added,
followed by vortexing at 4 ◦ C. The supernatant
representing the cytoplasmic plus periplasmic protein (CPP) fraction was saved after ultracentrifugation at 100 000 × g, as above. This fraction corresponds to the cytosol fraction according to Moreno
et al. (1985). Because the level of spheroplasting
is always maintained around 90%, the last pellet after isolation of the periplasmic fraction still
contains some periplasmic proteins. Thus, the estimated level of protein isolated in the PP fraction
is a lower estimate of the amount secreted into the
periplasmic fraction. Furthermore, it is difficult to
exactly measure how much of the CPP protein is
comprised by the periplasmic fraction, because it is
possible that some amount may remain in the cell,
even after processing of the secretory signal. The
insoluble pellet fraction, which corresponds to the
membrane fraction of Moreno et al. (1985), was
saved. In agreement with Moreno et al. (1985) we
found that most of the invertase activity fractionated in the periplasmic and the pellet (membrane)
fractions (data not shown). All the fractions were
stored at −70 ◦ C.
Western blot analysis and zymography
Protein extracts were subjected to SDS-polyacrylamide gel electrophoresis (PAGE; 12.5%) according to Laemmli (1970) and immunoblotted using
antibodies against SK. Polyclonal antibodies were
raised against SK by injecting the rabbits with the
SK band (expressed and secreted from the recombinant clone; Pratap et al., 1996) excised from
SDS–PAGE gels. Alkaline phosphatase-conjugated
anti-rabbit antibody was used for Western detection of SK (Promega). Biologically active SK bands
were identified by zymography after electrophoresis on 12.5% SDS–PAGE and overlaying with
soft agar mixed with plasminogen (Roche) and
skimmed milk, as described previously (Malke and
Ferretti, 1984; Pratap et al., 1996). The active SK
protein band was visualized as a clear band after
incubation at 37 ◦ C for 2–3 h.
Plate assay for SK
Yeast strains harbouring either the chimeric SK
plasmid (pJRK1) or control vector (pREP3) were
grown on a PMA leu− plate containing 25 µM
thiamine and incubated at 30 ◦ C for 2 days. Then
Copyright  2004 John Wiley & Sons, Ltd.
R. Kumar and J. Singh
they were replica-plated on to PMA leu− plates
(without thiamine) and overlaid with 10 ml of 1.2%
agarose in TE containing skimmed milk (10%)
and 50 µg plasminogen. The plates were further
incubated at 30 ◦ C and a halo was observed after
5–6 h (Pratap et al., 1996).
Estimation of plasminogen activator activity of
SK
Biologically active SK was quantitated essentially as described previously (Jackson et al.,
1981; Pratap et al., 1996) using Chromozyme
PL (Boehringer-Mannheim) as a synthetic substrate. Briefly, human plasminogen (BoehringerMannheim) was activated with SK at 37 ◦ C for
15 min and amidolytic activity of this complex was
monitored at 405 nm after the addition of synthetic
plasmin substrate, chromozyme PL. Purified SK
(World Health Organization) was used as a standard.
Generation of a mutant lacking extracellular
protease activity
Wild-type strains of Sz. pombe possess a potent
extracellular protease activity, as monitored by
development of a halo around the colony after
overnight growth (18–24 h) on a YEA plate containing skimmed milk or longer on a synthetic
plate containing skimmed milk. Such a wild-type
strain (SP837) was mutagenized with EMS and
spread on YEA plates. Nearly 10 000 individual
colonies were picked and spotted on to several
plates (250–500/plate) containing skimmed milk
and incubated at 30 ◦ C. Colonies lacking any halo
were picked up and subjected to secondary screening, after which only one mutant lacking a halo
was identified.
Treatment of cell extracts with PNGase F
(Peptide N-glycosidase F)
Treatment with PNGase F was carried out as
described by Maley et al. (1989). 10 µg RNAse B
(Sigma) and 40 µg protein extract were denatured
at 100 ◦ C for 10 min in the denaturing buffer (0.5%
SDS, 1% β-mercaptoethanol) separately. 1/10 volume each of 0.05 M sodium phosphate buffer (pH
7.5, 25 ◦ C) and 10% NP-40 were added. To these
reactions, 5 µl PNGase F (500 000 U/ml; New
England Biolabs) was added and, after incubation
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Expression of streptokinase in Sz. pombe
at 37 ◦ C for 8–10 h, the samples were subjected to
electrophoresis.
Treatment of cell extracts with Endo H
Extracts were subjected to treatment with Endo H
according to Maley et al. (1989). 10 µg RNAse B
(Sigma) and 40 µg protein extract were denatured
at 100 ◦ C for 10 min in the denaturing buffer
(0.5% SDS, 1% β-mercaptoethanol, 50 mM sodium
citrate, pH 5.5, at 25 ◦ C). To the reaction 5 µl Endo
H (500 000 U/ml; NEB) was added and incubation
continued at 37 ◦ C for 8–10 h. The treated samples
were subjected to SDS–PAGE and Coomassie
staining.
PAS Staining for glycoproteins
The in-gel PAS staining assay for checking the
carbohydrate modification of streptokinase was
carried out as described by Segrest and Jackson
(1972). Samples were subjected to electrophoresis
and the gel was soaked sequentially in 7.5%
acetic acid at room temperature for 1 h and 0.2%
aqueous periodic acid at 4 ◦ C for 45 min. They
were then immediately placed in Schiff’s reagent
and refrigerated for 45 min. The gel was destained
in two or three changes of a solution of 10% acetic
acid. For faster destaining, the gel was treated with
a solution prepared by adding 50 ml 1 N HCl and
5.0 g potassium metabisulphite in 950 ml water.
To prepare Schiff’s reagent, 0.5 g basic fuchsin
was dissolved in 100 ml boiling water. The mixture
was stirred for 5 min and cooled to 50 ◦ C. It was
then filtered and 10 ml 1 N HCl was added to the
filtrate. The solution was cooled to 25 ◦ C and 0.5 g
potassium metabisulphite added. The solution was
allowed to stand in dark for 24 h.
Results
Construction of the expression vector pJRK1
There is only one recent report of use of the secretory signal from Sz. pombe to facilitate the processing and secretion of the heterologous protein (GigaHama, 1997). Therefore, we proposed to express
streptokinase gene by attaching a secretory signal
unique to Sz. pombe to facilitate its secretion. The
plasmid pREP3, containing the thiamine-regulated
Copyright  2004 John Wiley & Sons, Ltd.
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promoter nmt1, was used as the backbone vector (Forsburg, 1993; Maundrell, 1993). It contains
the 2.2 kb HindIII fragment of LEU2 of S. cerevisiae as a selectable marker and 1.2 kb EcoRI
fragment containing ars1 of Sz. pombe to help
maintain a high copy number in the transformants
(Moreno et al. 1993). In addition, it contains the
1.2 kb nmt1 promoter and the nmt1 transcription
termination signal. A fusion between the signal
sequence of the Plus pheromone of Sz. pombe (Imai
and Yamamoto, 1994) and the mature SK protein
of Streptococcus (Malke and Ferretti, 1984) was
generated (Figure 1A) by bridge overlap extension
PCR (Mehta and Singh, 1999) and cloned between
the Xho1 and BamH1 sites of the plasmid pREP3
to yield the plasmid construct pJRK1 (Figure 1B).
Optimization of expression of the chimeric SK
in Sz. pombe
Plasminogen clearing plate assay was performed
with the transformants harbouring the plasmid
pJRK1 to assess the level of SK activity. Two
independent transformants, clone 1 and clone 2,
of the Sz. pombe strain SP837 produced a clear
zone when grown on plates containing skimmed
milk and plasminogen, while the same strain containing the control vector showed no clear zone
(Figure 2A), indicating that the recombinant plasmid pJRK1 expresses SK activity in Sz. pombe and
secretes it into the medium.
Preliminary studies to achieve conditions for
optimum expression of SK showed that growth in
non-selective YEA medium, followed by induction by transfer to PMA leu− medium lacking
thiamine, yielded better expression than growth
in PMA leu− medium plus thiamine followed by
induction by transfer to PMA leu− medium lacking
thiamine. It is possible that a low, leaky expression, even in the presence of thiamine (which
is documented and also observed by us; Forsburg, 1993), combined with slower growth in synthetic medium, may reduce the expression level by
putting a metabolic load on cells. Therefore, cells
containing pJRK1 were grown in YEA medium
to different OD600 (0.2–1.5) and then inoculated
into synthetic PMA leu− medium lacking thiamine
to induce SK expression. Surprisingly, while the
result of the plate assay suggests secretion of SK
into the medium (Figure 2A), no SK activity could
be detected in the culture medium (not shown),
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R. Kumar and J. Singh
Figure 2. Expression of recombinant plasmid pJRK1 carrying the chimeric construct PSK in Sz. pombe yields a functional
streptokinase. (A) Halo assay: cells from fresh cultures of the wild-type Sz. pombe strains carrying vector alone and two
independent clones of PSK (chimera of Plus pheromone and authentic form of streptokinase) (pJRK1 clones 1 and 2) were
spotted on a PMA leu− plate (I) and on a PMA leu− plate containing skimmed milk (II). The plates were incubated at 30 ◦ C
for 2 days and monitored for halo. (B, C) Time course of induction of expression of streptokinase in wild-type Sz. pombe
strains followed by Coomassie staining (B) and Western blot analysis (C). Whole cell extracts were prepared from cells
containing vector pREP3 alone (lanes 1 and 2) or plasmid pJRK1 (lanes 3–9) and grown in the presence (lanes 2 and 3) or
absence (lanes 1, 4–9) of thiamine. Samples containing pJRK1 were grown to OD600 = 0.2 (lane 4), 0.3 (lane 5), 0.4 (lane
6), 0.5 (lane 7), 0.7 (lane 8) and 0.9 (lane 9) and then grown in the absence of thiamine for 15 h before preparation of the
extracts. The extracts were resolved by electrophoresis in duplicate on 12.5% SDS–polyacrylamide gels and either stained
with Coomassie blue (B) or subjected to Western blotting using anti-streptokinase polyclonal antibodies raised in rabbits
(C). The precursor (49.5 kDa) and mature form of SK (47 kDa) are indicated by arrows. (D) Zymographic analysis of the
extracts of wild-type cells expressing control vector pREP3 (lane 1) or pJRK1 (lane 3) and epp1 mutant cells expressing
pJRK1 (lane 2). The control SK produced by a recombinant E. coli strain is shown in lane 4
indicating that it is not secreted into the medium
under the conditions of growth in culture conditions (this point is further addressed below). However, two faint bands of 49.5 and 47 kDa could
be observed in Coomassie-stained gels of whole
cell extracts prepared from different cultures with
starting OD600 = 0.2 (arrows in Figure 2B, lane
4), which persisted with increase in the starting
OD600 (Figure 2B, lanes 5–8). These results were
paralleled by those of immunoblotting with antiSK antibody (Figure 2C, lanes 4–8). However,
Copyright  2004 John Wiley & Sons, Ltd.
no corresponding bands were observed in extracts
prepared from samples with starting OD600 = 0.9
(Figure 2B, C, lane 9) or higher (not shown).
The SK activity in the extract was quantitated
using chromozyme substrate and a maximum level
of specific activity of 1580 IU/mg protein was
observed with starting OD600 = 0.7 (Table 1), representing mid- to late log phase of growth. However, at starting OD600 = 0.9, there was a sudden loss of SK activity, as measured by activity (Table 1) and protein (Figure 2D, lane 9). The
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Expression of streptokinase in Sz. pombe
Table 1. Specific activity of SK achieved after growth at
different OD600 at the time of induction
OD600
0.2
0.3
0.4
0.5
0.7
0.9
SK activity
(IU/ml)
Total protein
(mg/ml)
Specific activity
(IU/mg)
700 ± 12
1767 ± 18
412 ± 24
2133 ± 16
2450 ± 10
0
1.95 ± 0.08
3.6 ± 0.15
0.9 ± 0.08
1.65 ± 0.10
1.55 ± 0.09
1.77 ± 0.08
358.9 ± 20
491 ± 15
457 ± 14
1293 ± 64
1581 ± 80
0
OD405 of sample × Std. SK
× 100 × dilution
SK activity (IU/ml) =
OD405 of Std. SK
factor.
0 denotes no detectable activity.
maximum level of activity achieved in Sz. pombe
was found to be comparable to that of SK expressed
in E. coli (1500 IU/ml; Pratap et al., 1996) and
P. pastoris (3200 IU/ml; Pratap et al., 2000). Densitometric scanning of Coomassie-stained gels indicated that the levels of SK expression was 1–2% of
total cellular proteins and total yield was approximately 24.5 mg/l of culture.
Streptokinase expressed in Sz. pombe is
functionally active
To check whether the SK expressed in Sz. pombe is
functionally active, we performed the in situ plasminogen clearing assay after resolving the protein
extracts by SDS–PAGE. Interestingly, clear zones
representing the plasminogen clearing activity were
observed, which co-migrated with the stained 49.5
and 47 kDa bands (Figure 2D, lane 3). Thus, both
forms of SK are functional. The lower band comigrated with the mature SK band obtained from a
recombinant clone expressed in E. coli (Figure 2D,
lane 4), suggesting that the 49.5 kDa band may correspond to the chimeric protein that included the
Plus pheromone signal, while the lower band of
47 kDa may correspond to the mature SK lacking
the signal sequence (also see below).
Effect of recombinant SK expression on
growth rate
To check whether the expression of SK might exert
any deleterious or toxic effect on the host cells,
the growth rate of cells expressing the plasmid
pJRK1 in presence and absence of thiamine were
compared. We found that under both conditions,
cells exhibited similar rates of growth up to 5 h
Copyright  2004 John Wiley & Sons, Ltd.
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after transfer to PMA leu− medium. However, cells
grown in the absence of thiamine showed a slower
growth rate than those grown in the presence of
thiamine (Figure 3) at the subsequent time points.
From the slopes of the curves in the log phase, the
generation time was calculated to be approximately
6 and 14 h for cells harbouring the recombinant
plasmid pJRK1 and grown in the presence and
absence of thiamine, respectively. The growth rate
of cells harbouring the control vector was similar
in the presence and absence of thiamine as well as
to the cells harbouring pJRK1 in the presence of
thiamine (not shown). Thus, expression of the SK
recombinant plasmid causes nearly 50% decrease
in the growth rate of the cells.
To check whether this reduction in the growth
rate might be correlated with the expression of SK,
the SK expression level was monitored by Western
blotting at different time points of induction. The
results show that expression of SK starts at 6 h
of induction, keeps on accumulating up to 15 h
(Figure 3B) and declines sharply thereafter. These
data indicate a correlation between the time of
onset of expression of SK (Figure 3B) with a
decrease in the growth rate of the cells expressing
SK (Figure 3A), suggesting that the expression
of SK is not toxic to the cells but exerts a
metabolic load on the host cells, leading to a
reduction in their growth rate. This interpretation
is supported by our observation that, when grown
in the presence of thiamine, the cells harbouring
the plasmid pJRK1 exhibit the same growth rate
as those with control vector pREP3, as mentioned
above (data not shown). However, the time of
maximum induction at 15 h and complete lack of
expression after 18 h after induction is in complete
contrast with the nmt1 promoter, which is known
to bring about maximum level of expression 18 h
after induction (Moreno et al., 1991). We speculate
that this difference may be because of the induction
regime: while generally the pre-induction growth
is normally carried out in synthetic and selective
medium containing thiamine, here it was carried
out in rich, non-selective, YEA medium. Further,
we find that, surprisingly, even in the rich, nonselective medium there is no significant loss of
plasmid (more than 98% cells retain the plasmid;
data not shown). Thus, the induction regime used
by us, which has been found to yield higher
expression levels, may somehow reduce the time
required for achieving the maximum levels of
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R. Kumar and J. Singh
2
A
Growth OD600nm
1.5
1
0.5
+ Thiamine
− Thiamine
0
0
4
8
12
16
20
Time (hrs)
B
0
3
6
9
12
15
18
KDa
49.5
Time of
Induction
(hrs)
47.0
1
2
3
4
5
6
7
Figure 3. Expression of SK has a moderately deleterious effect on the growth rate of the host Sz. pombe cells. (A) Growth
rate of wild-type strain expressing control vector and plasmid pJRK1. Cells were grown in YEA and then transferred to
PMA leu− medium lacking thiamine, as described in Materials and methods. Aliquots were withdrawn at indicated time
points up to 15 h, the OD600 recorded and plotted. (B) Extracts were prepared from cells induced for 0 (lane 1), 3 (lane
2), 6 (lane 3), 9 (lane 4), 12 (lane 5), 15 (lane 6) and 18 h (lane 7) were subjected to immunoblotting with anti-SK antibody
induction, although the reason for complete loss of
expression after 18 h of induction is not clear. We
speculate that this may be because of proteloytic
degradation or loss of solubility of the protein.
Generation of a mutant defective in the
extracellular protease activity in Sz. pombe,
epp1
Earlier, we observed that Sz. pombe possesses
a potent extracellular protease activity, as monitored by formation of a halo upon growth of
its colonies on YEA plates containing skimmed
milk (Figure 4A, top). Since this activity could
potentially cause the degradation of the SK that is
secreted into the periplasmic fraction (see below),
we carried out EMS mutagenesis of Sz. pombe
cells, and isolated one mutant that did not produce a halo when grown on skimmed milk plates
Copyright  2004 John Wiley & Sons, Ltd.
(Figure 4A, bottom). In a genetic cross with a wildtype strain, this mutation segregated in a 2+:2−
ratio in all the 30 tetrads dissected. This indicates that the phenotype results from a mutation
in a single locus. Interestingly, the mutant was
also found to be temperature-sensitive for growth
at 36 ◦ C, while it grows normally at 25 ◦ C and
30 ◦ C. More interestingly, the mutant also exhibited a defect in mating type silencing (not shown).
Thus, the gene corresponding to the mutation has
pleiotropic effects on cellular growth, gene silencing and proteolytic pathway. This is not altogether
surprising, since mutations in the Rhp6-dependent,
ubiquitin-mediated proteolytic pathway have also
been shown to affect growth rate and silencing
in Sz. pombe (Singh et al., 1998). The genomic
clone complementing the mutation reveals several ORFs, including a hypothetical protein with
unknown function (Kumar and Singh, unpublished
Yeast 2004; 21: 1343–1358.
Expression of streptokinase in Sz. pombe
1351
Figure 4. The putative protease mutant epp1 is defective in extracellular protease activity and achieves higher level of
expression under stationary phase conditions (A) and distribution of SK activity in different cellular fractions of Sz. pombe
harbouring the plasmids pREP3 and pJRK1 (B). (A) Wild-type (top) and epp1 mutant (bottom) were grown in YEA plates
containing skimmed milk. The wild-type strain produces a halo (top) while the epp1 mutant does not (bottom). (B) For cell
fractionation, an overnight culture of Sz. pombe was inoculated into 20 ml YEA at OD600 = 0.1 and incubated at 30 ◦ C for
5–6 h (until OD600 = 0.7). The cells were collected by centrifugation, washed with sterile PMA leu− , resuspended in PMA
leu− and further incubated at 30 ◦ C for 15 h for induction. The periplasmic protein (PP) and cytoplasmic (CPP) fractions
were prepared and subjected to electrophoresis. Lanes 1–3, vector control; lane 1, PP fraction; lane 2, CPP fraction; lane
3, pellet fraction; lane 4, SK from E. coli; lanes 5–7 correspond to PP (lane 5), CPP (lane 6) and pellet (lane 7) fraction
of wild-type cells and lanes 8–10 correspond to PP (lane 8), CPP (lane 9) and pellet (lane 10) fractions of epp1 mutant
expressing pJRK1 in medium lacking thiamine. A smudge around the 50 kDa position in lane 8 is an artifact of Western
blotting and does not represent a real band. (C) Expression of SK in the epp1 mutant in which expression of SK was
induced at starting OD600 = 0.9. The lanes indicate the periplasmic (PP), cytoplasmic (CPP) and pellet fractions of epp1
mutant strain carrying the control plasmid pREP3 and SK plasmid pJRK1. WCL denotes whole cell extract
results). The actual gene assignment awaits further
experiments. Although the exact gene function is
not known, it is tentatively named as extracellular
protease of pombe (epp1 ). We envisaged that such
a mutant would be useful in minimizing the degradation of the secreted streptokinase.
Copyright  2004 John Wiley & Sons, Ltd.
Fractionation of the cell extracts from the
wild-type and epp1 mutant cells expressing
pJRK1
To ascertain whether the 47 kDa band observed in
earlier experiments (Figure 2B, C) is the result of
processing by the secretory pathway, and also to
Yeast 2004; 21: 1343–1358.
1352
check whether the expression in the epp1 mutant
might aid in reducing the already negligible level
of degradation, we prepared the cytoplasmic plus
periplasmic (CPP) and periplasmic (PP) fractions
of wild-type and epp1 mutant strains expressing
plasmid pJRK1 (in separate experiments, we found
that no detectable SK protein or activity was
secreted into the culture medium; data not shown).
We used the assay for invertase, a periplasmic
enzyme in Sz. pombe (Moreno et al., 1985), to
validate the fractionation protocol. We found that
most of the invertase activity fractionated in the
periplasmic and pellet (membrane) fractions but not
the cytosol (CPP), which is in agreement with the
results of Moreno et al. (1985) and validates our
fractionation procedure (data not shown). As shown
in Figure 4B, the PP fraction in both wild-type and
epp1 mutant extracts contained only the 47 kDa
band and no trace of a 49.5 kDa band (Figure 4B,
lanes 5 and 8; the smudge seen around 50 kDa in
lane 8 is an artifact of Western blotting), while the
CPP fraction showed both of the bands (Figure 4B,
lanes 6 and 9). A substantial fraction ( 50%) of
both bands was not extractable and was found to be
associated with the pellet fraction (Figure 4B, lanes
7 and 10). Thus, the 47 kDa band is very likely
yielded by processing via the secretory pathway
and export into the periplasmic fraction. Notably,
a smaller band of 45 kDa, apparently resulting
from partial proteolysis, was also observed in the
PP fraction from the wild-type cells (Figure 4B,
lane 5) but not in the epp1 mutant (Figure 4B,
lane 8). These results suggest that the putative
epp1 gene product may be responsible, directly or
indirectly, for the partial degradation of the mature
SK found in the periplasmic fraction in wild-type
cells. This inference is supported by the results of
in situ activity assay of the protein expressed in the
epp1 mutant, which does not indicate any trace of
proteolytic products (Figure 2D, lane 2), while in
case of the wild-type host cells, a faint smear below
the 45 kDa band (Figure 2D, lane 3) is indicative of
a low level of degradation in the wild-type strain.
The origin of the doublet at the position of the
47 kDa bands in lanes 6, 8 and 9 (Figure 4B) is
not clear. However, this doublet is not observed
reproducibly and may be an experimental artifact
of band splitting caused by presence of an abundant
protein located at the same position, as is obvious
from Coomassie staining (Figure 2B).
Copyright  2004 John Wiley & Sons, Ltd.
R. Kumar and J. Singh
Optimization of SK expression in the epp1
mutant
The loss of expression of SK at starting OD600 =
0.9 in wild-type cells is not paradoxical, as it
has also been observed in case of secreted protein SEAP under stationary phase conditions (Sambamurti, 1997). We believe that this could be
because the putative protease is also secreted maximally under stationary phase conditions (see Materials and methods; not shown). If so, the epp1
mutant should exhibit detectable expression even
at starting OD600 = 0.9 and above. Interestingly,
we find that the epp1 mutant exhibits a high level
of expression even up to starting OD600 = 1.2
(Table 2), with the peak of activity being achieved
at OD600 = 9, which is much higher than that
achieved with wild-type strain (Table 1). However, at starting OD600 = 1.5, there was a complete loss of SK expression (Table 2) in the epp1
mutant as well. It is possible that apart from the
Epp1 product there may be another protease(s) that
degrades the SK. Alternatively, the cells expressing SK may ultimately die abruptly, due to excessive metabolic load followed by degradation of the
endogenous SK.
Quantitation of data showed that a higher level of
SK activity was secreted into the periplasmic fraction at starting OD600 = 0.9 than at OD600 = 0.7
in epp1 mutant (cf. Tables 3 and 4) which, in
turn, was higher than wild-type at OD600 = 0.7
(Tables 3 and 4). Surprisingly and most interestingly, however, the SK produced in the epp1
mutant at starting OD600 = 0.9 was completely in
Table 2. Specific activity of SK achieved after growth at
different starting OD600 at the time of induction in the
epp1 mutant
OD600
0.2
0.3
0.4
0.5
0.7
0.9
1.2
1.5
SK activity
(IU/ml)
Total protein
(mg/ml)
Specific activity
(IU/mg)
781 ± 9
1116 ± 14
1367 ± 17
2250 ± 20
3333 ± 22
4133 ± 30
3033 ± 28
0
1.96 ± 0.08
2.05 ± 0.11
1.94 ± 0.08
1.62 ± 0.08
1.66 ± 0.08
1.68 ± 0.09
2.08 ± 0.11
2.26 ± 0.10
399 ± 12
542 ± 23
704 ± 22
1389 ± 59
2008 ± 80
2460 ± 110
1458 ± 63
0
OD405 of sample × Std. SK
× 100 × dilution
SK activity (IU/ml) =
OD405 of Std. SK
factor.
0 denotes no detectable activity.
Yeast 2004; 21: 1343–1358.
Expression of streptokinase in Sz. pombe
Table 3. SK activity in different cellular fractions
prepared from wild-type and epp1 mutant strains of
Sz. pombe expressing the plasmid pJRK1 after growth up to
OD600 = 0.7 and 15 h of induction
Cellular
fraction
activity∗
SK
(IU/ml)
In wild type:
PP
1200 ± 12
CPP
1329 ± 15
In epp1 mutant:
PP
1329 ± 14
CPP
1243 ± 10
Total protein
(mg/ml)
Specific activity
(units/mg)
1.48 ± 0.06
1.74 ± 0.08
811 ± 24
764 ± 25
1.12 ± 0.05
1.41 ± 0.07
1187 ± 40
882 ± 37
PP, periplasmic proteins; CPP, cytoplasmic plus periplasmic proteins.
∗ SK activity was not determined in pellet.
Table 4. SK activity in different cellular fractions prepared
from epp1 mutant strain of Sz. pombe expressing the plasmid
pJRK1 after growth up to OD600 = 0.9 and 15 h of induction
Cellular
fraction
PP
CPP
SK activity∗
(IU/ml)
Total protein
(mg/ml)
Specific activity
(units/mg)
1867 ± 25
1867 ± 35
1.26 ± 0.11
1.48 ± 0.15
1482 ± 100
1261 ± 96
PP, periplasmic proteins; CPP, cytoplasmic plus periplasmic proteins.
∗ SK activity was not determined in pellet.
the 47 kDa form and fractionated in the periplasmic
fraction (Figure 4C, lane 4), and the CPP fraction (Figure 4C, lane 5), with no detectable amount
in the pellet fraction (Figure 4C, lane 6). In contrast, as shown above, at starting OD600 = 0.7,
roughly equal amounts of precursor and processed
forms of SK were produced, both in the pellet and
cytoplasmic fraction (Figure 4B, lanes 9 and 10).
Thus, induction of expression following an initial
growth up to OD600 = 0.9 not only improves the
extractability but also facilitates the complete processing of the secretory signal. The improved level
of processing and extractability of SK expressed
at starting OD600 = 0.9 (Figure 4C) as compared
to OD600 = 0.7 (Figure 4B) is interesting and surprising. Although the reason for this is not clear,
it is possible that the extent of processing and
secretion with respect to protein expression may
improve with progression of growth or with the
altered cellular physiology during late log phase.
Alternatively, there may be lag during growth of
cells expressing SK between the expression of SK
and its folding, which improves its processing and
extractability.
Copyright  2004 John Wiley & Sons, Ltd.
1353
Lack of post-translational SK modification in
Sz. pombe
Since N-linked glycosylation is known to occur in
Sz. pombe (Giga-Hama, 1997a), we checked the
possibility that the 49.5 kDa band may represent
the glycosylated form of SK. Therefore, we treated
the extract prepared from cells expressing pJRK1
with PNGaseF (NEB), an amidase which cleaves
between the innermost GlcNAc and asparagine
residues of the high mannose, hybrid and complex oligosaccharides from N-linked glycoproteins
(Chu, 1986). N-glycosylated RNAse B was used
a control (Chu, 1986). As shown in Figure 5B,
PNGase F treatment caused a reduction in the size
of RNAse B (cf. lanes 5 and 6). However, no
change in the positions of the SK bands of 49.5
and 47 kDa was discernible between treated and
untreated samples (Figure 5A, lanes 1 and 2). Furthermore, PNGase F could deglycosylate RNAse B
even when mixed with extracts from cells expressing the plasmid pJRK1 (Figure 5B, lanes 3 and
4), indicating that PNGaseF is active in Sz. pombe
extracts. Similar results were obtained with endo
H, a glycosidase, which cleaves the chitobiose core
of high mannose and some hybrid oligosaccharides
from N-linked glycoproteins (Maley et al., 1989).
RNAse B could be deglycosylated by endo H to
a form with faster electrophoretic mobility, either
alone (Figure 5D, lanes 5 and 6) or when mixed
with cell extract (Figure 5D, lanes 3 and 4), but
did not affect the electrophoretic mobility of either
the 49.5 or 47 kDa forms of SK.
To directly assess the presence or absence of carbohydrate side chains, we used the PAS staining
method (Segrest and Jackson, 1972). While the glycoprotein RNAse B and some proteins in extracts
of cells expressing streptokinase both stained with
the PAS reagent (Figure 6A, lanes 2 and 3; note
the aberrant location of the band corresponding
to RNAse B, which, because of an electrophoresis artifact, seems to coincide with the marker
lane), no band co-migrating with the 49.5 and
47 kDa bands of streptokinase (Figure 6B, lane 2;
cf. Figure 6A, lane 3 — note solid arrows corresponding to the position of the 49.5 and 47 kDa
bands of SK) showed any staining with the PAS
reagent.
These results indicate a lack of N-linked glycosylation of SK and, together with the results
of Figure 4C and 2D, strongly suggest that the
Yeast 2004; 21: 1343–1358.
1354
R. Kumar and J. Singh
B
PNGase F
-
Ext
RNAse B
pJRK1
pJRK1 Ext
A
+
-
-
pJRK1 Ext
+
RNAse B
-
RNAse B
+
-
+
kD
49.5
47
(+CHO)
(-CHO)
1
C
2
3
4
5
6
RNAse B
-
Ext
pJRK1 Ext
D
pJRK1
Endo H
kD
1
2
+
-
-
pJRK1 Ext
+
RNAse
B
-
RNAse
B
-
+
+
49.5
47
(+CHO)
(-CHO)
1
2
1
2
3
4
5
6
Figure 5. SK expressed in Sz. pombe lacks N-linked glycosylation. 40 µg whole cell extract prepared from cells expressing
pKRK1 was resolved by SDS–PAGE followed by Western blotting with anti-SK antibody. (A, C) Lanes 1 and 2 represent
extracts that were either not treated or treated with PNGaseF (A) or Endo H (C). PNGase and Endo H are active in Sz.
pombe extracts (B and D, respectively). Lanes 1 and 2 contain protein extracts from cells expressing the plasmid pJRK1
and RNAse B, respectively. 10 µg RNAse B, either alone (lanes 5 and 6) or premixed with 40 µg Sz. pombe cell extract
(lanes 3 and 4) was either treated with PNGase F (lanes 4 and 6) (B), or Endo H (D) or mock-incubated (lanes 3 and 5) (B
and D). Following the treatment, the samples were subjected to SDS–PAGE and staining with Coomassie brilliant blue
49.5 and 47 kDa bands correspond to the Pfactor–mature SK fusion protein and the mature
SK, respectively, both of which lack any carbohydrate moiety. The co-migration of the 47 kDa
band of mature SK expressed and secreted by E.
coli (Pratap et al., 1996) with the band of the same
size in extracts of wild-type and epp1 mutant cells
expressing pJRK1 (Figure 2D, cf. lane 4 with lanes
2 and 3) also argues strongly against any modification and in favour of correct processing of
the P-factor signal sequence to yield the mature
Copyright  2004 John Wiley & Sons, Ltd.
SK, which is secreted into the periplasmic fraction (Figure 4B and 4C). A confirmation of correct
processing of the secretory signal, however, awaits
N-terminal sequencing of the 49.5 and 47 kDa
bands. Conventional chromatographic approaches
used for purifying SK secreted from E. coli or
Streptococcus cells expressing the gene were found
to be inadequate for purifying the SK from the
cell extracts of Sz. pombe cells because of cofractionation of a few proteins of similar size with
SK through all the chromatographic steps. Work
Yeast 2004; 21: 1343–1358.
pJRK1 ext.
B
Marker
pJRK1 ext.
kD
Marker
A
1355
RNAse B
Expression of streptokinase in Sz. pombe
kD
175
kD
175
83
62
83
47.5
62
33
49.5
47
47.5
RNAse B
1
2
3
1
2
Figure 6. SK expressed in Sz. pombe lacks carbohydrate side chains. (A) RNAse B (lane 2) and whole cell extract
prepared from cells of Sz. pombe expressing the plasmid pJRK1 (lane 3) were subjected to SDS–PAGE followed by
periodic acid–Schiff (PAS) staining. Because of aberrant migration during electrophoresis, bands in lanes 2 (RNAse B) and 3
(Sz. pombe proteins) show a curvature to the left side, resulting in appearance of RNAse B of lane 2 (indicated by a line
arrow) close to the marker lane (lane 1). (B) Western blotting shows the position of the 49.5 and 47 kDa forms of SK (lane
2). Lane 1 shows the markers (A and B). The position of the 49.5 and 47 kDa bands of SK is indicated in the PAS-stained
gel with solid arrows, showing a lack of reaction with PAS dye
is in progress to devise alternative strategies to
purify SK bands and carry out their N-terminal
mapping.
Discussion
Streptokinase is an important thrombolytic drug
for therapeutic purposes. Expression of SK has
been attempted in different heterologous systems.
However, when expressed in E. coli, nearly 30%
of the secreted SK is degraded (Pratap and Dikshit, 1998), while in P. pastoris, the secreted SK
is heavily glycosylated (Pratap et al., 2000). We
have attempted to express and secrete a bacterial protein such as streptokinase in Sz. pombe by
attachment of a signal sequence of Plus pheromone
Copyright  2004 John Wiley & Sons, Ltd.
of Sz. pombe. A high level of expression comparable to that achieved earlier in E. coli and P.
pastoris, concomitantly with processing of the signal and secretion of nearly 50% of mature SK
into the periplasmic fraction (but not into the culture medium), was observed. The estimated amount
secreted into the periplasmic fraction is likely to be
an underestimate because the extent of spheroplasting carried out in the fractionation experiments was
kept around 90%, as discussed in Materials and
methods. However, interestingly, only the 47 kDa
band was detected in the PP fraction, while both
the 49.5 and 47 kDa bands were observed in the
CPP fraction, a result consistent with the possibility of the 49.5 kDa band representing the P-factor
signal sequence–SK chimera and the 47 kDa band,
the mature SK lacking the P factor signal sequence.
Yeast 2004; 21: 1343–1358.
1356
A lack of secretion of SK in to the growth
medium upon expression in Sz. pombe, despite the
production of a halo in the plasminogen plate assay,
is surprising. It may be because the SK may be
localized both in the periplasmic fraction and on
the outside surface of the cell wall but fails to be
exit the cell wall into the medium. A similar result
was obtained with the endogenous secretory protein invertase from Sz. pombe, which was shown
to exist in the periplasmic and membrane fractions but failed to be secreted into the medium
(Moreno et al., 1985). Another possibility is that
SK may be secreted in to the medium but might
be degraded by the extracellular protease. This
possibility is supported by the reported degradation of the mouse α-amylase by a chymostatinsensitive protease activity (which is secreted into
the medium), when expressed and secreted in Sz.
pombe using the killer toxin secretory signal (Tokunaga et al. 1993). It remains to be checked whether
the activity affected by epp1 mutation is also sensitive to chymostatin. Another possibility that may
explain the discrepancy is that the presence of the
substrates (skimmed milk and plasminogen) within
the plates may stimulate the secretion of SK in the
in situ plate assay, but their absence in the liquid
culture medium may not stimulate the release of
SK. Moreover, in the liquid medium as well as in
the plate assay, the protease is secreted only after
16–18 h. In the plate assay the SK is expressed
and secreted within 5–6 h and may escape degradation by the protease because of presence of large
excess of plasmin substrate (skimmed milk) and
also the lag in the appearance of protease, but in
liquid culture SK expression occurs at a slower rate
(15 h of induction), coincidentally with the appearance of protease activity in the medium which may
degrade the SK in the medium.
There have been several studies showing secretion of proteins in Sz. pombe using heterologous
secretory signals, e.g. human antithrombin III (Broker et al., 1987), human gastric lipase (Smerdon
et al., 1995) and human alkaline phosphatase (Sambamurti, 1997) and S. cerevisiae invertase (Sanchez
et al., 1988; Zarate and Belda, 1996). Recently,
the homologous secretory signals of P factor have
also been used for expression and secretion of IL-6
(Giga-Hama, 1997b). Although an efficient secretion was achieved in Sz. pombe, the secreted IL-6
was highly glycosylated and also showed a significant level of degradation (Giga-Hama, 1997b).
Copyright  2004 John Wiley & Sons, Ltd.
R. Kumar and J. Singh
Thus, this is the first time that expression and secretion of SK into the periplasmic fraction has been
achieved in Sz. pombe using a pheromone signal.
The fact that the SK expressed in Sz. pombe does
not show any modifications is both interesting and
surprising. Expression of SK in P. pastoris was
recently shown to result in significant glycosylation
(Pratap et al., 2000), which is consistent with presence of several consensus sites for glycosylation in
the SK sequence (Pratap et al., 2000). Furthermore,
expression of IL-6 using a similar secretory signal
in Sz. pombe also yielded extensively glycosylated
IL-6 (Giga-Hama, 1997b). A likely explanation is
that glycosylation occurs at the time of secretion
during IL-6 expression in Sz. pombe (Giga-Hama,
1997b) and SK expression in P. pastoris (Pratap
et al., 2000).
What factors are responsible for secretion of proteins into the medium vs. periplasmic fraction is not
understood. However, very few proteins in S. cerevisiae are known to be exported into the medium
as compared to the periplasmic fraction (GigaHama, 1997a). The α-factor pheromone, which is
widely used for expression and secretion of several heterologous proteins in S. cerevisiae, is synthesized as a precursor, which is proteolysed by
the Kex2 protease to yield the functional polypeptide. This polypeptide is kept unfolded in the cytoplasm by chaperones and subsequently translocated
across the endoplasmic reticular membrane into the
lumen. No major studies on the secretory pathway in Sz. pombe have been reported. However,
the Plus factor is also synthesized as a precursor of four repeats and a signal sequence, which
is proteolysed by the signal peptidase to yield the
functional pheromone (Imai and Yamamoto, 1994).
The mechanism of the processing is not known,
although the Sxa2 protease is considered to be the
likely site-specific protease that cleaves the precursor polypeptide into the functional pheromone
peptides (Imai and Yamamoto, 1994). However, it
is only recently that these homologous sequence
elements are beginning to be exploited as secretory
signals to facilitate the secretion of the heterologous proteins in Sz. pombe (Giga-Hama, 1997b;
this study).
We anticipated that the secreted heterologous
protein is likely to be subject to degradation by
the proteases that are also secreted. Therefore, we
assayed for and discovered an unknown potent
extracellular protease activity in Sz. pombe. Using
Yeast 2004; 21: 1343–1358.
Expression of streptokinase in Sz. pombe
EMS mutagenesis, we were able to obtain one
mutant epp1, which shows a drastic reduction
in the extracellular protease activity. A particularly interesting aspect of this study is that when
SK was expressed in the epp1 mutant, not only
was a higher level of expression achieved but it
occurred with a greater degree of conversion of
the expressed SK into the mature SK, enhanced
extractability and lack of degradation. Thus, the
epp1 mutation has paradoxical effects: on the one
hand, it abolishes the degradation of SK secreted
into the periplasmic fraction, while on the other
hand it enhances the processing of the Plus-factor
signal sequence–SK fusion into the mature SK. It
is possible that the epp1 gene plays a role in the
secretory pathway and the epp1 mutation may have
subtle and apparently contradictory effects on the
protein degradation and processing related to secretion. Another interesting characteristic of the epp1
mutant is the high level of SK expression under stationary phase conditions, while in wild-type cells
the expression peaks only during mid-log phase and
declines sharply in the mid- to late log phase. This
is consistent with the observation that the extracellular protease activity is maximally observed in
wild-type cells under late log-stationary phase conditions (data not shown). Work is in progress to
identify the epp1 gene.
In conclusion, the main advantageous features of
this study are three-fold. First, the nmt1 promoter
provides for regulated expression of the heterologous gene. Second, we have achieved expression,
processing and secretion of the mature SK without
any glycosylation and degradation; these features
are advantageous because the modified forms may
not be desirable for therapeutic purposes, as they
may elicit an immune response and the degraded
forms would then have to removed by cumbersome
procedures. Third, using the epp1 mutant, a higher
level of expression of mature, undegraded and
unmodified SK, with its secretion almost entirely
into the periplasmic fraction, could be achieved.
Because of these features, this system of expression of SK may be well suited for commercial
exploitation. The main drawback of this study is
that secretion of mature SK into the extracellular
medium has not been achieved. We have generated hypersecretory mutants with higher levels of
SK activity, as monitored by halo assay. Further
work will focus on screening these mutants as well
Copyright  2004 John Wiley & Sons, Ltd.
1357
as the epp1 mutant for secretion of SK into the
extracellular medium.
Acknowledgements
We gratefully acknowledge the suggestions of V. Vyas, K.
L. Dikshit and G. Sahni. We thank V. Vyas for providing
the SK protein from E. coli for raising antibodies.
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