Secretion signal for protein expression
Materials and methods plates containing 0.2% glucose or at increasing dosage of
Construction of secretory CAT and β-galactosidase expres- arabinose. The expression and secretion of functional SS-β-
sion vectors lactamase was visualized as colony formation.
For liquid assay, 5 ml of RM medium (1 M9 salts, 2%
The isolation and initial cloning of SS into pEGFP-N1, to
casamino acids, 0.2% D-glucose, 1 mM MgCl2, 50 µg/ml
yield pSSEGFP was described in Tan et al. (Tan et al.,
kanamycin) was inoculated with either a single recombinant
2000a). Detailed sequences and cloning strategies of secretory
or untransformed LM194 colony. The induction procedure
chloramphenicol acetyltransferase (SSCAT) and β-galactosid-
was as described by the manufacturer (Invitrogen). Prior to
ase (SS-Gal) can be obtained from the corresponding author.
induction, a 1 ml aliquot of culture was removed, processed
The vector maps of various constructs are illustrated in Figure 1.
and designated the zero time point. The medium was clariﬁed
Cell culture and transfections off bacteria by centrifugation and sterile ﬁltered using a
COS-1, NIH/3T3, HeLa and CHO-B cells were maintained in 0.22 µm membrane. The periplasmic space fraction was
DMEM while EPC and CHSE were cultured in MEM. All isolated from the cell lysate (Laforet and Kendall, 1991). Both
media were phenol-red free and supplemented with 10% the medium and periplasmic fraction were assayed for
charcoal/dextran-treated fetal bovine serum. Cells were trans- β-lactamase activity (Cohenford et al., 1988).
fected with 1 µg of SS-fusion construct:control vector in a
ratio of 8:2, by lipofectamine (Gibco BRL) as described by Results
For estrogen-induction experiment, cells were co-transfected SS directs the secretion of reporter protein into culture medium
with ERU-psp-SSCAT, pSGcER (chicken estrogen receptor) To investigate whether SS can direct the secretion of common
and pSEAP-Control as described in Tan et al. (Tan et al., reporter proteins from various eukaryotic hosts, fusion con-
1999). For studies on the rate of secretion, better comparison structs of SS with CAT and β-galactosidase driven by
between the CAT and β-galactosidase ELISA were achieved constitutive CMV or SV40 promoter, were transfected into a
by adapting to ﬂuorescence assays using the DIG Fluorescence variety of cell lines, namely COS-1, NIH/3T3, CHO-B, EPC,
Detection ELISA (Boehringer Mannheim). SEAP was deter- HeLa and CHSE. As illustrated in Figure 2, SSCAT, ssEGFP
mined ﬂuorimetrically (LS-50B, Perkin Elmer) at Ex360nm and SS-Gal were effectively secreted and accumulated in the
and Em449nm. SSCAT and SS-Gal were detected at Ex440nm culture medium of all the cell lines tested, although the amount
and Em550nm. of SS-fusion protein produced varied. Despite being diluted
Expression of SSCAT in S.cerevisiae in the culture medium, the secreted recombinant proteins were
detectable within 24 h, indicating high level expression.
The construct pYEX-SSCAT was transformed into S.cerevisiae
DY150 (Chen et al., 1992). The transformants were selected Rapid secretion rate of SSCAT and SS-Gal compared to
on synthetic minimal medium (MM) agar containing all the SEAP
required supplements except uracil. For expression analysis, a The amount of SS-fusion protein secreted at various time
100 ml YEPD medium (2% yeast extract, 1% mycopeptone, intervals was determined using a standard curve generated
2% D-glucose, 5 HTA: 100 mg each of histidine, tryptophan from the positive control provided by the kits. The rate of
and adenine, pH 5.0) was inoculated with a single clone and secretion was determined by the gradient of the best-ﬁt line
grown for 16 h at 30°C. Subsequently, 50 ml of the yeast when the amount of secreted protein was plotted against time.
cultures were grown independently for 72 h in 2 1 l bafﬂed The mean rates of secretion of SSCAT and SS-Gal were
ﬂasks containing either 200 ml of YEPD or MM. At indicated 15.8 fg/ml SSCAT/ng β-galactosidase/min 0.12 fg/ml/min
time intervals, 2 ml aliquots of culture were centrifuged to and 12.1 fg/ml SS-Gal/ng SEAP/min 0.09 fg/ml/min,
obtain yeast pellet and culture supernatant. The yeast cells respectively (Figure 3). In comparison, SEAP was secreted
were lysed with glass beads (Roopashree et al., 1996), whereas at a rate of 4.76 fg/ml SEAP/ng β-galactosidase/min
the culture medium was collected and frozen without any pre- 0.06 fg/ml/min. The rate of SSCAT and SS-Gal secretion was
treatment. The pH of the culture was adjusted to pH 5.0 almost 3-fold higher than SEAP. Thus, this indicates that there
using 1 M potassium phosphate buffer (pH 8.0). SSCAT was is a rapid post-translational processing of the SS.
measured as described above. Inducible expression of SSCAT protein correlated with its
Arabinose-induced expression of modiﬁed SS-β-lactamase in mRNA level
bacteria The expression and secretion of SS-fusion proteins, in particular
Transformants of E.coli LM194 with pBADSSblactKana were SSCAT, were also examined using an inducible promoter. The
selected for by plating on LB agar containing 50 µg/ml estrogen-induced expression and secretion of SSCAT was
kanamycin. For the ampicillin plate assay, LM194 competent observed in all the cell lines tested, although the amount of
bacteria were transformed and plated on ampicillin LB agar SSCAT produced varied. Uninduced COS-1 cells exhibited
Fig. 1. (a) The diagrammatic map of the pSSCAT vector. The expression of the SSCAT gene is driven by a strong constitutive promoter, CMV. The start
ATG codon of the CAT gene was mutated to CTG to ensure efﬁcient translation initiation at SS. (b) pSS-Gal construct map. pSS-Gal used the backbone from
the β-Gal-promoter (Clontech) except that SS was subcloned in-frame upstream of the β-galactosidase gene. (c) psp-SSCAT map. The psp-SSCAT harbors the
secreted SSCAT. The multiple cloning site (MCS) is as illustrated. (d) Map of the ERU-psp-SSCAT construct. The ERU-psp-SSCAT is similar to the
psp-SSCAT except that the 565 bp ERU of Xenopus vitellogenin B1 gene is subcloned upstream of the SV40 promoter. (e) pYEX-SSCAT vector map.
The pYEX-SSCAT is the yeast vector expressing SSCAT. The vector backbone is pYEX-S1. The original K.lactis signal peptide was replaced by SS.
(f) pBADSSblactKana vector map. The mutant β-lactamase gene, whose secretion is directed by SS is subcloned into the vector backbone of pBAD vector
(Invitrogen). The SS-β-lactamase insert is regulated by the araBAD promoter. Another selective antibiotic resistance gene (kanamycin from pGFP-N3) was
used to replace the parental ampicillin resistance gene of pBAD vector.
N.S.Tan, B.Ho and J.L.Ding
Fig. 3. Rate of secretion of SSCAT and SS-Gal in comparison with SEAP.
COS-1 cells were transfected with SS-fusion construct:control vectors. After
36 h post-transfection, at intervals of 15 min over a period of 2 h, the
medium from cells of each time point was removed and replaced with 1 ml
of fresh medium. After the last time point, which should represent 0 min, an
additional 1 h incubation was employed for all cultures to avoid low
reading variations. At the end of incubation, the medium was clariﬁed via
centrifugation. The rate of secretion was determined by the gradient of best-
ﬁt line when the amount of secreted protein was plotted against time. The
values for SSCAT and SEAP secreted were normalized by β-galactosidase
production. Similarly, the values for secreted SS-Gal were normalized with
medium is directly proportional to changes in intracellular
concentration of SSCAT mRNA, a northern kinetic analysis
was performed under estrogen-stimulation. Figure 4c indicates
that the level of SSCAT protein secreted into the culture
medium was directly proportional to changes in intracellular
concentration of SSCAT mRNA. The results indicate that the
Fig. 2. (a) Western blot analysis of SSEGFP expression in COS-1 cells. The previously observed rapid secretion of SS-fusion proteins
majority of SSEGFP was secreted into the culture medium. This shows that driven by constitutive promoters is not due to the strength of
SS can direct secretion of a reporter gene, EGFP. For each sample, 30 µg of the promoter, but rather, the properties of SS as a secretion
total protein from culture medium was used for electrophoresis. Lanes: M,
molecular weight marker; 1, untransfected COS-1 cell culture medium,
signal. Thus far, we have demonstrated that SS is functional
24 h; 2, culture medium, 24 h post-transfection; 3, culture medium, 48 h in several common higher eukaryotic hosts, both mammalian
post-transfection (b) Western blot analysis of SS-Gal using mouse anti-β- and non-mammalian. In addition, under similar experimental
galactosidase. Fifty micrograms of culture medium was loaded and conditions, a higher level of SS-fusion proteins was detected
electrophoresed in a 10% SDS–PAGE. Lanes: 1, molecular weight marker; in the extracellular medium as compared to SEAP.
2, day 5 medium; 3, day 4 medium; 4, day 3 medium; 5, day 2 medium;
6, control medium; 7, 20 µg of cell lysate from day 5 culture. The western The novel SS can direct secretion of recombinant proteins in
blot was developed using goat anti-mouse-HRP and chemiluminescent yeast
substrate. (c) Secreted SSCAT expression was observed in all the six cell
lines tested (COS-1, NIH/3T3, CHO-B, EPC, HeLa, CHSE). SSCAT was Although, recombinant protein expression in yeast has its
measured using ELISA. Values represent the mean of four independent limitations, it is still a favorable choice because it can be
experiments. cultivated readily in large-scale fermentation, with an advant-
age of releasing relatively little extraneous protein material into
only marginal increase in SSCAT over a period of 24 h. For the medium and post-translational modiﬁcations of proteins. To
induced COS-1 cells, the increase in SSCAT can be detected further examine the versatility of SS, the secretion of SS-
as early as 2 h, reaching a peak of 7-fold increase at 12 h fusion protein, SSCAT, driven by constitutive PGK promoter
post-induction (Figure 4a). Estrogen-induced expression of was studied in two independent S.cerevsiae transformants
SSCAT can also be observed in other vertebrate cell lines, cultured in two different media.
namely NIH/3T3, CHO-B and EPC cells (Figure 4b). The SSCAT expression proﬁle was monitored over 72 h in
To verify that the level of secreted SSCAT in the culture yeast grown in YEPD (rich medium) and MM (minimal
Secretion signal for protein expression
Fig. 4. (a) Inducible expression and secretion of the recombinant CAT reporter. COS-1 cells were cotransfected with ERU-psp-SSCAT, pSGcER and pSEAP-
Control. Estrogen-induced expression of SSCAT was monitored over a period of 24 h upon addition of 10–7 M of E2. (b) Estrogen-inducibility observed in
other eukaryotic cells. SSCAT was produced and secreted into the culture medium by NIH/3T3, CHO-B and EPC. Values are means of four independent
experiments. (c) Northern blot analysis of E2-induced SSCAT expression for ERU-psp-SSCAT. The levels of SSCAT secreted into the culture medium are
directly proportional to changes in intracellular concentration of SSCAT mRNA. Actin was used to normalize the result.
medium). After 24–48 h of culture, the yeast transformants increase in secreted SSCAT. This effect is less pronounced in
grown in MM secreted signiﬁcantly less SSCAT in the medium. the rich YEPD medium, probably because it supports high-
It is unlikely that the overall SSCAT expression was reduced density growth and has higher buffering capacity. The amount
in MM-cultured yeast since comparable SSCAT protein was of SSCAT detected in both types of culture media was
observed in the yeast lysate of both MM and YEPD cultures. comparable at 72 h (Figure 5).
Interestingly, the decrease corresponds to a drop in the pH of It is noteworthy that although the amount of SSCAT in
MM. Adjusting the pH back to 5, resulted in a tremendous the medium is ~50% that of yeast lysate, this is likely an
N.S.Tan, B.Ho and J.L.Ding
under-representation of the secreted SSCAT. The amount of
periplasmic SSCAT was not determined, but was instead
included in the values of SSCAT in the yeast lysate.
The growth and expression proﬁles of SS-β-lactamase in
Ampicillin which belongs to the β-lactam group of antibiotics,
binds to and inhibits a number of enzymes in the bacterial
membrane that are involved in the synthesis of the cell wall
(Waxman and Strominger, 1983). The ampicillin resistance
gene codes for β-lactamase, and is secreted into the periplasmic
space of the bacterium, where it catalyzes hydrolysis of the
β-lactam ring, with concomitant detoxiﬁcation of the drug
(Sykes and Mathew, 1976). As such, this imposes an absolute
requirement on the bacteria for both high level and rapid
expression/secretion of functional β-lactamase to ensure its
survival. We next sought to investigate if SS can fulﬁll these
requirements necessary for the growth of the bacterial host.
To this end, we have constructed a mutant β-lactamase, SS-
β-lactamase, where its native secretion signal was replaced by
SS in a construct, pBADSSblactKana. The expression of
Fig. 5. Expression proﬁle of SSCAT in two different yeast transformants.
Secretion of SSCAT into culture medium is signiﬁcantly higher in the rich
YEPD medium. It is important to note that the cell lysate, in this instance,
refers to SSCAT in both the cytosol and periplasmic space. Consequently,
secretion of SSCAT is more efﬁcient than that reﬂected by SSCAT detected
in the medium only.
Fig. 6. (a) Plate assay for SS-β-lactamase. A functional kanamycin gene
was demonstrated by the ability of the bacteria to grow on kanamycin-
containing LB agar. No bacterial colonies were observed for either 0.2%
glucose or 0.0002% arabinose. As the concentration of arabinose inducer
was increased, smaller bacterial colonies were observed. (b) SS-β-lactamase
expression proﬁle in culture medium. Transformants induced with 0.0002%
arabinose displayed the highest level of SS-β-lactamase in the medium.
(c) SS-β-lactamase accumulation in the periplasmic space. Accumulation of
SS-β-lactamase in the periplasmic space displayed inducer dose-dependent
expression. Rapid and high accumulation of SS-β-lactamase in the
periplasmic space does not necessarily translate into higher amounts of
recombinant protein in the culture medium.
Secretion signal for protein expression
Table I. Comparison of efﬁcacy of SS with other secretion signals in four common expression hosts
Secretion signals Bacteriaa Yeastb Insectc Mammalian References
SS Current work; Tan et al., 2000a,b; Wang et al., 2001
Growth hormone Gray et al., 1985; Asakura et al., 1995
Serum albumin Coloma et al., 1992; Kirkpatrick et al., 1995
Human placental alkaline phosphatase Golden et al., 1998
Staphylococcal protein A Uhlen and Abrahmsen, 1989; Allet et al., 1997
Honeybee melittin Tessier et al., 1991
Ecdysteroid UDP-glucosyltransferase Laukkanen et al., 1996
Tissue plasminogen activator Farrell et al., 1999
α-Mating factor Brake et al., 1984; Kjeldsen, 2000
PHO1 Laroche et al., 1994
K.lactis killer toxin Baldari et al., 1987
OmpA/T Pines and Inouye, 1999
Haemolysin Blight and Holland, 1994; Chervaux et al., 1995
Bacteriophage fd gene III Rapoza and Webster, 1993
, secretion competency of recombinant proteins from the host.
aIncludes both Gram-positive and -negative bacteria.
bIncludes S.cerevisiae, Schizosaccharomyces pombe and P.pastoris.
cIncludes lepidoteran (i.e. baculovirus system) and Drosophila.
the SS-β-lactamase came under the control of an inducible (iii) the expression and secretion of the gene products must
arabinose-responsive promoter. As illustrated in Figure 6a, no be of appreciable quantity and functional. Currently, no single
colonies were seen in the absence or presence of 0.0002% secretion signal has been demonstrated to be effective in
arabinose or 0.2% glucose alone, whereas dose-dependent both prokaryotic and eukaryotic host expression systems. The
arabinose-induced (0.002–0.2%) expression and secretion of currently available secretion signals have exhibited limited
SS-β-lactamase permitted the bacterial transformants to survive functionality and/or non-compatibility for cross-host recombin-
on ampicillin LB agar plates. Interestingly, the colony size ant protein expression (Table I). Therefore, availability of a
appeared distinctively smaller with increasing levels of ara- common broad-host secretion signal is highly desirable. The
binose. major objective of this study was to evaluate the efﬁciency of
To further examine the efﬁcacy of SS directed β-lactamase SS in directing cross-host expression and secretion of foreign
secretion, we decided to measure SS-β-lactamase activities via proteins. Consequently, SS was subcloned upstream of three
a liquid assay. The pBADSSblactKana clone grown in RM reporter protein genes—EGFP, CAT and β-galactosidase. These
medium with 0.2% glucose (i.e. no induction) exhibited a three proteins were chosen because of their different size and
similar growth proﬁle as the control LM194 host bacteria (data origin (prokaryotic versus eukaryotic).
not shown). Concomitant with the plate assay, no SS-β- Based on strict deﬁnition, no functional heterologous secre-
lactamase activity can be detected in the culture medium and tion signal was reported for bacterial use. Although the
periplasmic space (Figure 6b and c) of uninduced trans- expression and secretion of numerous heterologous genes,
formants. The addition of arabinose resulted in expression and such as human superoxide dismutase (Takahara et al., 1988),
accumulation of SS-β-lactamase in the culture medium and have been successful in bacteria, most if not all bacterial
periplasmic space. Even more surprising is that the highest expression utilized secretion signals of prokaryotic origin
level of enzyme was detected when 0.0002% arabinose was (Table I). Perhaps, the closest example was that of human
used (Figure 6c). This apparent conﬂict was due to the growth- growth hormone (hGH). Gray et al. (Gray et al., 1985)
inhibitory effect on the bacteria when induced at a high compared the efﬁciency of export of hGH directed by either
concentration of arabinose (data not shown). The dose-depend- its own signal sequence or the E.coli Pho A signal sequence.
ent expression proﬁle of SS-β-lactamase, however, was not Results indicated that the secretions are comparable with 72%
observed after 4 h. Similar results were obtained using the of the hGH localized in the periplasm. However, the efﬁcacy
TOP10 strain of E.coli, although the overall protein expression of the hGH signal in directing the secretion of heterologous
level decreased by ~20% (data not shown). proteins in bacteria has not been reported. In comparison, the
potential of SS to direct secretion of proteins in E.coli was
Discussion evaluated by the secretion of a modiﬁed SS-β-lactamase. Via
The fundamental basis for the search of a cross-host secretion plate and liquid assays, we showed that the secretion is rapid
signal really lies in the efﬁcacy between heterologous versus and at least 50% of the protein is detectable in the extracellular
homologous secretion signals. Heterologous secretion signals medium upon induction. However, high doses of the inducer,
refer to the use of this signal for the secretion of heterologous arabinose, led to lower secreted product. Overloading the
gene products, as well as from a different host from which export machinery may result from inefﬁcient secretion of a
the signal sequence was derived. In contrast, homologous foreign protein because the protein is expressed at levels that
secretion signals refer to the secretion of its natural gene simply exceed cellular capacity. This is the ﬁrst report of a
product from the same host species. Certainly, a cross-host functional heterologous signal sequence in bacteria that permits
secretion signal will have to satisfy three other important appreciable yield of secreted recombinant protein.
criteria: (i) this signal must confer secretion to gene products The ﬁrst heterologous secretion signal for yeast was the
of different origins (prokaryotic or eukaryotic); (ii) the func- human serum albumin (hHSA). This human secretion signal
tionality of this signal must extend beyond its original host; works well in yeast, producing ~50% of the hHSA in yeast
N.S.Tan, B.Ho and J.L.Ding
fermentation media (Sleep et al., 1990). This signal results not This study reports the identiﬁcation and development of a
only in the hHSA secretion but also the secretion and desired cross-host secretion signal. Its ability to direct recombinant
processing of other heterologous genes, such as human protein secretion was evaluated with SS-fusion reporter proteins
immunodeﬁciency virus (HIV) gp120 (Lasky et al., 1986) and in various hosts—higher eukaryote, yeast and bacteria. We
somatostatin (Itakura et al., 1977). Again, the functionality envision that fusion of the SS to recombinant genes will prove
of hHSA signal in bacteria was not reported. Interestingly, to be a valuable tool for efﬁcient protein secretion in a broad
expression of hGH in yeast results in properly processed hGH heterologous host expression system. This secretion signal can
in yeast media, suggesting that the signal recognition is not be incorporated into the ‘donor vector’ of various multi-vector
ﬂawed. However, only 10% of the expressed protein is secreted cloning systems, such as Gateway™ (Gibco BRL) and Echo™
whereas 90% of hGH remains cell-associated and retains the Cloning (Invitrogen), which can then be transferred into
entire signal sequence (Hitzeman et al., 1984). In comparison, various host expression vectors for expression and secretion
we used SS to direct the secretion of a prokaryotic protein, of recombinant proteins. This secretion signal can also be
SSCAT. As shown in Figure 5, at least 50% of the protein incorporated into various reporter assay systems for rapid, and
was secreted into the yeast media. Unlike, the hHSA signal minimal set-up reporter gene analyses. While an exhaustive
sequence, SS is applicable in bacteria. It is worth noting that screen of all proteins is beyond the scope of this study, during
in rapidly growing expression hosts, such as that of E.coli and the process of preparing this paper, the SS has been further
S.cerevisiae, the rate of secretion is greatly inﬂuenced by their evaluated by other researchers and was proven to yield varied
growth conditions. Consequently, for optimal secretion of success in the secretion of other recombinant proteins, for
recombinant protein via SS, in rapidly growing expression example, in Drosophila S2 cells, Sf9 cells (Wang et al., 2001)
hosts, a compromise must be struck between growth condition and E.coli (unpublished data).
and concentration of the inducer, in order to regulate the rate
of recombinant protein production and its secretion. Acknowledgements
Many secreted eukaryotic proteins are efﬁciently processed We thank Professor W.Wahli for Xenopus Vtg B1 ERU, and Professor
in mammalian expression host via their native signal sequences. P.Chambon for pSG cER. This work was funded by NUS Grant
Hence, a more comprehensive study was done with SS. The RP3999900/A and NSTB Grant LS/99/004.
SS is able to direct secretion of both prokaryotic (SSCAT and
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Received May 25, 2001; revised December 18, 2001; accepted January 4, 2002