SlideShare ist ein Scribd-Unternehmen logo
1 von 9
Downloaden Sie, um offline zu lesen
Protein Engineering vol.15 no.4 pp.337–345, 2002


Engineering a novel secretion signal for cross-host recombinant
protein expression




Nguan Soon Tan1,2, Bow Ho3 and Jeak Ling Ding1,4                           tailoring to meet the stringent requirements for each protein
1Department                             3Department                        product to ensure correct folding, activity and desired yield.
            of Biological Sciences and             of Microbiology,
National University of Singapore, Singapore 117543                         Furthermore, the flexibility of a common secretion signal
4To
                                                                           sequence with which to secrete a wide variety of heterologous
    whom correspondence should be addressed at: Department of Biological
Sciences, National University of Singapore, 10 Kent Ridge Crescent,
                                                                           fusion proteins from various hosts into the extracellular medium
Science Drive 4, Singapore 117543.                                         is not available.
E-mail: dbsdjl@nus.edu.sg                                                     Protein secretion is one of the most important issues of
2Present address: Institut de Biologie Animale, Batiment de Biologie,      protein expression in fundamental processes of living cells.
Universite de Lausanne, CH-1015, Lausanne, Switzerland                     Successful protein secretion requires effective translocation of
Protein secretion is conferred by a hydrophobic secretion                  the protein across the endoplasmic recticulum or plasma
signal usually located at the N-terminal of the polypeptide.               membrane. Proteins destined for secretion are targeted to the
We report here, the identification of a novel secretion signal              membrane via their respective secretion signals that are usually
(SS) that is capable of directing the secretion of recombinant             located at the N-terminal of nascent polypeptides. These signals
proteins from both prokaryotes and eukaryotes. Secretion                   display very little primary sequence conservation. However,
of fusion reporter proteins was demonstrated in Escherichia                they all possess three general domains: an N-terminal region
coli, Saccharomyces cerevisiae and six different eukaryotic                that varies widely in length, but typically, contain amino acids
cells. Estrogen-inducibility and secretion of fusion reporter              which contribute a net positive charge to this domain; a central
protein was demonstrated in six common eukaryotic cell                     hydrophobic region made up of a block of seven to 16
lines. The rate of protein secretion is rapid and its expres-              hydrophobic amino acids; and a C-terminal region that includes
sion profile closely reflects its intracellular concentration                the signal cleavage site (Nothwehr and Gordon, 1990; Pines
of mRNA. In bacteria and yeast, protein secretion directed                 and Inouye, 1999). Since the principles of protein transloca-
                                                                           tion mechanism are evolutionarily conserved (Schatz and
by SS is dependent on the growth culture condition and
                                                                           Dobberstein, 1996), it is conceivable that there exists a secretion
rate of induction. This secretion signal allows a flexible
                                                                           signal that is operational in both prokaryote and eukaryote,
strategy for the production and secretion of recombinant
                                                                           viz, cross-host.
proteins in numerous hosts, and to conveniently and rapidly
                                                                              Our previous efforts to express and secrete the limulus
study protein expression.
                                                                           Factor C, a highly complex serine protease, in Escherichia
Keywords: broad hosts/protein expression/secretion signal
                                                                           coli, Pichia pastoris and COS cells using its native hydrophobic
                                                                           signal, Saccharomyces cerevisiae α-mating factor or
                                                                           Kluyveromyces lactis killer toxin secretion signal were unsuc-
Introduction
                                                                           cessful (Roopashree et al., 1995, 1996; and unpublished data).
With innovative genomics technology, genes are being disco-                Surprisingly, the secretion of the similar construct was achieved
vered faster than their functions can be characterized. As we              by a novel 15-residue hydrophobic secretion signal (SS) in
enter the era of proteomics, the ability to rapidly produce large          Drosophila S2 cells (Tan et al., 2000a). Furthermore, varying
numbers of proteins in a parallel manner becomes increasingly              the genes in the fusion or the tags, did not affect the high-
important. Determining their functions requires numerous                   level secretion and cleavage at the correct site (Tan et al.,
biophysical (e.g. crystallization, NMR, MS) and functional                 2000a,b). Despite the origin of this signal, 80% of the
studies (e.g. protein–protein interactions), each of which uses            recombinant proteins expressed by the heterologous insect host
a different expression vector. Hindrances to these analyses                were localized in the extracellular medium. In this study,
include the arduous task of subcloning, problems with reading              we demonstrate the versatility and functionality of SS for
frame and Kozak sequence, as well as the downstream                        recombinant protein expression and secretion in cross-hosts
purification protocols. A versatile system for transferring DNA             [E.coli, S.cerevisiae, higher eukaryotic cells—African green
fragments between vectors using the Cre-lox recombinase                    monkey kidney cells (COS-1), Chinese hamster ovary cells
technology has been recently developed (Liu et al., 1998). In              (CHO-B), fibroblast cells derived from Swiss mouse embryo
addition, the Sindbis expression system enables the rapid,                 (NIH/3T3), human cervical adenocarcinoma cells (HeLa), carp
high-level expression of heterologous proteins in a variety of             epithelial cells (EPC) and chinook salmon embryonic cells
eukaryotic cell lines derived from mammalian, avian and insect             (CHSE)]. The expression and secretion of the recombinant
hosts (Xiong et al., 1989). Recombinant proteins synthesized               proteins were performed using either a constitutive or an
in heterologous hosts may accumulate in one of three ‘compart-             inducible promoter. In addition, we compared the secretion
ments’: the cytoplasm, the periplasm or the extracellular                  rate of reporter protein directed by SS and human secreted
medium. Many overexpressed proteins from various origins                   alkaline phosphatase (SEAP) signal, and assessed the efficiency
have been purified from each of these locations. Whenever                   of secretion in different yeast media. This paper illustrates the
possible, secretion is the preferred strategy since it permits             engineering of SS to aid the production of secreted recombinant
easy and efficient purification from the extracellular medium.               protein for easier analysis. To the best of our knowledge, this
However, to date, each expression system needs specific                     study documents the only known cross-host secretion signal.
© Oxford University Press                                                                                                                 337
N.S.Tan, B.Ho and J.L.Ding




338
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
the manufacturer.
   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.

                                                                                                                                                  339
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
                                                                              SEAP.


                                                                              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
340
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
                                                                                                                                                  341
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
bacteria
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.

342
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
                                                                                                                                                     343
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
SS-β-galactosidase) and eukaryotic proteins (EGFP), regardless       References
of protein size. Moreover, the rate of secretion of heterologous     Allet,B., Bernard,A.R., Hochmann,A., Rohrbach,E., Graber,P., Magnenat,E.,
proteins is at least 3-fold faster than the SEAP native signal          Mazzei,G.J. and Bernasconi,L. (1997) Protein Expr. Purif., 9, 61–68.
                                                                     Asakura,A., Minami,M. and Ota,Y. (1995) Biosci. Biotechnol. Biochem., 59,
sequence. Taken together, SS is the only signal sequence                1976–1978.
known to date that is functional in all four common expression       Baldari,C., Murray,J.A., Ghiara,P., Cesareni,G. and Galeotti,C.L. (1987) EMBO
hosts (Table I).                                                        J., 6, 229–234.
   What makes SS such an efficient universal secretion signal?        Blight,M.A. and Holland,I.B. (1994) Trends Biotechnol., 12,450–455.
                                                                     Brake,A.J., Merryweather,J.P., Coit,D.G., Heberlein,U.A., Masiarz,F.R.,
SS is capable of cross-host secretion for several reasons. First,       Mullenbach,G.T., Urdea,M.S., Valenzuela,P. and Barr,P.J. (1984) Proc. Natl
it has the three domains typified in all secretion signals and           Acad. Sci. USA, 81, 4642–4646.
the presence of small amino acid residues at position –1 and –       Chen,D.C., Yang,B.C. and Kuo,T.T. (1992) Curr. Genet., 21, 83–84.
3 of the cleavage site (Jain et al., 1994). Secondly, the charge     Chervaux,C., Sauvonnet,N., Le Clainche,A., Kenny,B., Hung,A.L., Broome-
to hydrophobicity ratio between the N-terminal domain and               Smith,J.K. and Holland,I.B. (1995) Mol. Gen. Genet., 249, 237–245.
                                                                     Cohenford,M.A., Abraham,J. and Medeiros,A.A. (1988) Anal. Biochem., 168,
hydrophobic core, which is important for directing the protein          252–258.
to the membrane (Rusch et al., 1994; Izard et al., 1996),            Coloma,M.J., Hastings,A., Wims,L.A. and Morrison,S.L. (1992) J. Immunol.
represents a compromise of the requirements needed by both              Methods, 152, 89–104.
the prokaryotic and eukaryotic hosts. Lastly, while many             Farrell,P.J., Behie,L.A. and Iatrou,K. (1999) Biotechnol. Bioeng., 64, 426–433.
                                                                     Golden,A., Austen,D.A., van Schravendijk,M.R., Sullivan,B.J., Kawasaki,E.S.
currently available secretion expression vectors also satisfy           and Osburne,M.S. (1998) Protein Expr. Purif., 14, 8–12.
the first criteria, few possess the optimal ratio with respect to     Gray,G.L., Baldridge,J.S., McKeown,K.S., Heyneker,H.L. and Chang,C.N.
criteria two, and none of them address the issue of sequences           (1985) Gene, 39, 247–254.
beyond the cleavage site, i.e. C-terminal, necessary for effective   Hitzeman,R.A., Chen,C.Y., Hagie,F.E., Lugovoy,J.M. and Singh,A. (1984) In
and homogenous cleavage and thus secretion. It is conceivable           ed. Arthur P.Bollon Recombinant DNA Products: Insulin, Interferon and
                                                                        Growth Hormone. CRC Press, Boca Raton, FL.
that the C-terminal sequences are able to tolerate more degener-     Itakura,K., Hirose,T., Crea,R., Riggs,A.D., Heyneker,H.L., Bolivar,F. and
acy and hence the apparent redundancy to highlight this                 Boyer,H.W. (1977) Science, 198, 1056–1063.
criteria. Effective cross-host secretion, in our case, requires      Izard,J.W., Rusch,S.L. and Kendall,D.A. (1996) J. Biol. Chem., 271, 21579–
this neglected criterion to be resolved. Initial attempts to            21582.
                                                                     Jain,R.G., Rusch,S.L. and Kendall,D.A. (1994) J. Biol. Chem., 269, 16305–
reduce and/or remove the post-cleavage remnant residues                 16310.
resulted in non-secreted recombinant protein (unpublished            Kirkpatrick,R.B., Ganguly,S., Angelichio,M., Griego,S., Shatzman,A.,
data). This compromise of post-cleavage six residues in the             Silverman,C. and Rosenberg,M. (1995) J. Biol. Chem., 270, 19800–19805.
recombinant proteins is highly unlikely to alter the recombinant     Kjeldsen,T. (2000) Appl. Microbiol. Biotechnol., 54, 277–286.
protein functions. Comparatively, the post-cleavage remnant          Laforet,G.A and Kendall,D.A. (1991) J. Biol. Chem., 266, 1326–1334.
                                                                     Laroche,Y., Storme,V., De Meutter,J., Messens,J. and Lauwereys,M. (1994)
residues of SS are significantly smaller than GST or GFP and             Biotechnology, 12, 1119–1124.
possess lesser charge than the 6 histidine tag. While this work      Lasky,L.A.,      Groopman,J.E.,     Fennie,C.W.,     Benz,P.M.,     Capon,D.J.,
clearly documents SS as a cross-host secretion signal, the              Dowbenko,D.J., Nakamura,G.R., Nunes,W.M., Renz,M.E. and Berman,P.W.
functionality of the secreted recombinant protein produced by           (1986) Science, 233, 290–212.
                                                                     Laukkanen,M.L., Oker-Blom,C. and Keinanen,K. (1996) Biochem. Biophys.
any particular host will also strongly depend on other factors          Res. Commun., 226, 755–761.
such as post-translational modifications and intrinsic properties     Liu,Q., Li,M.Z., Leibham,D., Cortez,D. and Elledge,S.J. (1998) Curr. Biol.,
of the protein to be expressed.                                         8, 1300–1309.

344
Secretion signal for protein expression


Nothwehr,S.F. and Gordon,J.I. (1990) BioEssays, 12, 479–484.
Pines,O. and Inouye,M. (1999) Mol. Biotechnol., 12, 25–34.
Rapoza,M.P. and Webster,R.E. (1993) J. Bacteriol., 175, 1856–1859.
Roopashree,S.D., Chai,C., Ho,B. and Ding,J.L. (1995) Biochem. Mol. Biol.
  Int., 35, 841–849.
Roopashree,S.D., Ho,B. and Ding,J.L. (1996) Mol. Marine Biol. Biotechnol.,
  5, 334–343.
Rusch,S.L., Chen,H., Izard,J.W. and Kendall,D.A. (1994) J. Cell Biochem.,
  55, 209–217.
Schatz,G. and Dobberstein,B. (1996) Science, 271, 1519–1526.
Sleep,D., Belfield,G.P. and Goodey,A.R. (1990) Biotechnology, 8, 42–46.
Sykes,R.B. and Mathew,M. (1976) J. Antimicrob. Chemother., 2, 115–157.
Takahara,M., Sagai,H., Inouye,S. and Inouye,M. (1988) Bio/Technology, 6,
  195–198.
Tan,N.S., Frecer,V., Lam,T.J. and Ding,J.L. (1999) Biochim. Biophys. Acta,
  1452, 103–120.
Tan,N.S., Ho,B. and Ding,J.L. (2000a) FASEB J., 14, 859–870.
Tan,N.S., Ng,P.M.L., Yau,Y.H., Chong,P.K.W., Ho,B. and Ding,J.L. (2000b)
  FASEB J., 14, 1801–1813.
Tessier,D.C., Thomas,D.Y., Khouri,H.E., Laliberte,F. and Vernet,T. (1991)
  Gene, 98, 177–183.
Uhlen,M. and Abrahmsen,L. (1989) Biochem. Soc. Trans., 17, 340–341.
Wang,J., Ho,B. and Ding,J.L. (2001) Biotechnol. Lett., 23, 71–76.
Waxman,D.J. and Strominger,J.L. (1983) Annu. Rev. Biochem., 52, 825–869.
Xiong,C., Levis,R., Shen,P., Schlesinger,S., Rice,C.M. and Huang,H.V. (1989)
  Science, 243, 1188–1191.

Received May 25, 2001; revised December 18, 2001; accepted January 4, 2002




                                                                                                                 345

Weitere ähnliche Inhalte

Was ist angesagt?

Mammalian & Bacterial Expression
Mammalian & Bacterial ExpressionMammalian & Bacterial Expression
Mammalian & Bacterial ExpressionJesús C. Morales
 
Experession vectores ppt bijan zare
Experession vectores ppt bijan zareExperession vectores ppt bijan zare
Experession vectores ppt bijan zarebijan zare
 
Manipulation of gene expression in prokaryotes
Manipulation of gene expression in prokaryotesManipulation of gene expression in prokaryotes
Manipulation of gene expression in prokaryotesSabahat Ali
 
Lecture 2a cosmids
Lecture 2a cosmidsLecture 2a cosmids
Lecture 2a cosmidsIshah Khaliq
 
Cloning Of Malaria Genes Using Perkisus Marinus
Cloning Of Malaria Genes Using Perkisus MarinusCloning Of Malaria Genes Using Perkisus Marinus
Cloning Of Malaria Genes Using Perkisus MarinusLeslie21211
 
Eukayotic expression - vimmi.
Eukayotic expression - vimmi.Eukayotic expression - vimmi.
Eukayotic expression - vimmi.Vimlesh Gupta
 
Expression systems
Expression systemsExpression systems
Expression systemsBruno Mmassy
 
gateway cloning
gateway cloning gateway cloning
gateway cloning Hamza Khan
 
Seminario biología molecular. Juan camilo Botero
Seminario biología molecular. Juan camilo BoteroSeminario biología molecular. Juan camilo Botero
Seminario biología molecular. Juan camilo BoteroCamilo Botero
 
Expression of recombinant proteins in mammalian cell lines
Expression of recombinant proteins in mammalian cell linesExpression of recombinant proteins in mammalian cell lines
Expression of recombinant proteins in mammalian cell linesSandeep Kumar
 
08 Kjm206 Expression Vector, Plasmid Vector
08 Kjm206 Expression Vector, Plasmid Vector08 Kjm206 Expression Vector, Plasmid Vector
08 Kjm206 Expression Vector, Plasmid VectorJeneesh Jose
 
1-s2.0-037811199390549I-main
1-s2.0-037811199390549I-main1-s2.0-037811199390549I-main
1-s2.0-037811199390549I-mainTeresa Zimny
 
Recombinant protein expression in E.coli
Recombinant protein expression in E.coliRecombinant protein expression in E.coli
Recombinant protein expression in E.coliajithnandanam
 
Expression systems
Expression systemsExpression systems
Expression systemsankit
 
Strategies for Recombinant protein production in E.coli
Strategies for Recombinant protein production in E.coliStrategies for Recombinant protein production in E.coli
Strategies for Recombinant protein production in E.coliUka Tarsadia University
 
Generation of MRP2 Efflux Transporter Knock-Out in HepaRG Cell Line
Generation of MRP2 Efflux Transporter Knock-Out in HepaRG Cell LineGeneration of MRP2 Efflux Transporter Knock-Out in HepaRG Cell Line
Generation of MRP2 Efflux Transporter Knock-Out in HepaRG Cell Linemdmitc
 

Was ist angesagt? (20)

Mammalian & Bacterial Expression
Mammalian & Bacterial ExpressionMammalian & Bacterial Expression
Mammalian & Bacterial Expression
 
Experession vectores ppt bijan zare
Experession vectores ppt bijan zareExperession vectores ppt bijan zare
Experession vectores ppt bijan zare
 
Manipulation of gene expression in prokaryotes
Manipulation of gene expression in prokaryotesManipulation of gene expression in prokaryotes
Manipulation of gene expression in prokaryotes
 
Lecture 2a cosmids
Lecture 2a cosmidsLecture 2a cosmids
Lecture 2a cosmids
 
Cloning Of Malaria Genes Using Perkisus Marinus
Cloning Of Malaria Genes Using Perkisus MarinusCloning Of Malaria Genes Using Perkisus Marinus
Cloning Of Malaria Genes Using Perkisus Marinus
 
Eukayotic expression - vimmi.
Eukayotic expression - vimmi.Eukayotic expression - vimmi.
Eukayotic expression - vimmi.
 
Expression systems
Expression systemsExpression systems
Expression systems
 
gateway cloning
gateway cloning gateway cloning
gateway cloning
 
Seminario biología molecular. Juan camilo Botero
Seminario biología molecular. Juan camilo BoteroSeminario biología molecular. Juan camilo Botero
Seminario biología molecular. Juan camilo Botero
 
Expression of recombinant proteins in mammalian cell lines
Expression of recombinant proteins in mammalian cell linesExpression of recombinant proteins in mammalian cell lines
Expression of recombinant proteins in mammalian cell lines
 
08 Kjm206 Expression Vector, Plasmid Vector
08 Kjm206 Expression Vector, Plasmid Vector08 Kjm206 Expression Vector, Plasmid Vector
08 Kjm206 Expression Vector, Plasmid Vector
 
1-s2.0-037811199390549I-main
1-s2.0-037811199390549I-main1-s2.0-037811199390549I-main
1-s2.0-037811199390549I-main
 
Recombinant protein expression in E.coli
Recombinant protein expression in E.coliRecombinant protein expression in E.coli
Recombinant protein expression in E.coli
 
Expression vectors
Expression vectorsExpression vectors
Expression vectors
 
Expression systems
Expression systemsExpression systems
Expression systems
 
MOLECULAR GENETICS
MOLECULAR GENETICSMOLECULAR GENETICS
MOLECULAR GENETICS
 
Strategies for Recombinant protein production in E.coli
Strategies for Recombinant protein production in E.coliStrategies for Recombinant protein production in E.coli
Strategies for Recombinant protein production in E.coli
 
Expression vectors
Expression vectorsExpression vectors
Expression vectors
 
Bacterial genetics
Bacterial genetics   Bacterial genetics
Bacterial genetics
 
Generation of MRP2 Efflux Transporter Knock-Out in HepaRG Cell Line
Generation of MRP2 Efflux Transporter Knock-Out in HepaRG Cell LineGeneration of MRP2 Efflux Transporter Knock-Out in HepaRG Cell Line
Generation of MRP2 Efflux Transporter Knock-Out in HepaRG Cell Line
 

Andere mochten auch

Karen Hatten Experiment II Final Report
Karen Hatten Experiment II Final ReportKaren Hatten Experiment II Final Report
Karen Hatten Experiment II Final ReportKaren Hatten
 
BIO Philadelphia yeast expression 2005
BIO Philadelphia yeast expression 2005BIO Philadelphia yeast expression 2005
BIO Philadelphia yeast expression 2005Stephen Berezenko
 
Cloning and Expression of recombinant Protein
Cloning and Expression of recombinant ProteinCloning and Expression of recombinant Protein
Cloning and Expression of recombinant ProteinGaurav Dwivedi
 
Recombinant protein expression and purification Lecture
Recombinant protein expression and purification LectureRecombinant protein expression and purification Lecture
Recombinant protein expression and purification Lecturetest
 

Andere mochten auch (7)

Karen Hatten Experiment II Final Report
Karen Hatten Experiment II Final ReportKaren Hatten Experiment II Final Report
Karen Hatten Experiment II Final Report
 
sample questions
sample questionssample questions
sample questions
 
BIO Philadelphia yeast expression 2005
BIO Philadelphia yeast expression 2005BIO Philadelphia yeast expression 2005
BIO Philadelphia yeast expression 2005
 
Cloning and Expression of recombinant Protein
Cloning and Expression of recombinant ProteinCloning and Expression of recombinant Protein
Cloning and Expression of recombinant Protein
 
Mdadar 3
Mdadar   3Mdadar   3
Mdadar 3
 
Lab Report #2
Lab Report #2Lab Report #2
Lab Report #2
 
Recombinant protein expression and purification Lecture
Recombinant protein expression and purification LectureRecombinant protein expression and purification Lecture
Recombinant protein expression and purification Lecture
 

Ähnlich wie rprotein3

Invivo protein synthesis
Invivo protein synthesis Invivo protein synthesis
Invivo protein synthesis Msc2021
 
The Importance Of Micrornas
The Importance Of MicrornasThe Importance Of Micrornas
The Importance Of MicrornasKimberly Thomas
 
Lab Differential Expression Differential gene expression provides th.pdf
 Lab Differential Expression Differential gene expression provides th.pdf Lab Differential Expression Differential gene expression provides th.pdf
Lab Differential Expression Differential gene expression provides th.pdfrita892197
 
Lab Differential Expression Differential gene expression provides .pdf
 Lab Differential Expression Differential gene expression provides .pdf Lab Differential Expression Differential gene expression provides .pdf
Lab Differential Expression Differential gene expression provides .pdfbasilpaul63
 
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE B.pdf
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE B.pdfONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE B.pdf
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE B.pdfamzonknr
 
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE BAC.pdf
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE BAC.pdfONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE BAC.pdf
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE BAC.pdfamzonknr
 
Abstract conference mbsmb 2009
Abstract conference mbsmb 2009Abstract conference mbsmb 2009
Abstract conference mbsmb 2009Norhafilda Ismail
 
00003 Jc Silva 2006 Mcp V5n4p589
00003 Jc Silva 2006 Mcp V5n4p58900003 Jc Silva 2006 Mcp V5n4p589
00003 Jc Silva 2006 Mcp V5n4p589jcruzsilva
 
ACR2016 Wermuth Exosome poster
ACR2016 Wermuth Exosome posterACR2016 Wermuth Exosome poster
ACR2016 Wermuth Exosome posterKellan Carney
 
Nuclear Transport And Its Effect On Breast Cancer Tumor Cells
Nuclear Transport And Its Effect On Breast Cancer Tumor CellsNuclear Transport And Its Effect On Breast Cancer Tumor Cells
Nuclear Transport And Its Effect On Breast Cancer Tumor CellsStephanie Clark
 
Genetic Dna And Bioinformatics ( Accession No. Xp Essay
Genetic Dna And Bioinformatics ( Accession No. Xp EssayGenetic Dna And Bioinformatics ( Accession No. Xp Essay
Genetic Dna And Bioinformatics ( Accession No. Xp EssayJessica Deakin
 
ISSCR-2014-poster
ISSCR-2014-posterISSCR-2014-poster
ISSCR-2014-posterAlicia Lee
 
Research Symposium Poster Draft
Research Symposium Poster DraftResearch Symposium Poster Draft
Research Symposium Poster DraftSara Nass
 
Seminario biología molecular María de los Ángeles Montoya
Seminario biología molecular María de los Ángeles MontoyaSeminario biología molecular María de los Ángeles Montoya
Seminario biología molecular María de los Ángeles MontoyaMaradelosngelesMonto1
 
A panel of recombinant monoclonal antibodies against zebrafish
A panel of recombinant monoclonal antibodies against zebrafishA panel of recombinant monoclonal antibodies against zebrafish
A panel of recombinant monoclonal antibodies against zebrafishShahnaz Yusaf
 

Ähnlich wie rprotein3 (20)

Invivo protein synthesis
Invivo protein synthesis Invivo protein synthesis
Invivo protein synthesis
 
The Importance Of Micrornas
The Importance Of MicrornasThe Importance Of Micrornas
The Importance Of Micrornas
 
Lab Differential Expression Differential gene expression provides th.pdf
 Lab Differential Expression Differential gene expression provides th.pdf Lab Differential Expression Differential gene expression provides th.pdf
Lab Differential Expression Differential gene expression provides th.pdf
 
Lab Differential Expression Differential gene expression provides .pdf
 Lab Differential Expression Differential gene expression provides .pdf Lab Differential Expression Differential gene expression provides .pdf
Lab Differential Expression Differential gene expression provides .pdf
 
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE B.pdf
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE B.pdfONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE B.pdf
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE B.pdf
 
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE BAC.pdf
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE BAC.pdfONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE BAC.pdf
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE BAC.pdf
 
Abstract conference mbsmb 2009
Abstract conference mbsmb 2009Abstract conference mbsmb 2009
Abstract conference mbsmb 2009
 
00003 Jc Silva 2006 Mcp V5n4p589
00003 Jc Silva 2006 Mcp V5n4p58900003 Jc Silva 2006 Mcp V5n4p589
00003 Jc Silva 2006 Mcp V5n4p589
 
ACR2016 Wermuth Exosome poster
ACR2016 Wermuth Exosome posterACR2016 Wermuth Exosome poster
ACR2016 Wermuth Exosome poster
 
Nuclear Transport And Its Effect On Breast Cancer Tumor Cells
Nuclear Transport And Its Effect On Breast Cancer Tumor CellsNuclear Transport And Its Effect On Breast Cancer Tumor Cells
Nuclear Transport And Its Effect On Breast Cancer Tumor Cells
 
Presentation final
Presentation finalPresentation final
Presentation final
 
Genetic Dna And Bioinformatics ( Accession No. Xp Essay
Genetic Dna And Bioinformatics ( Accession No. Xp EssayGenetic Dna And Bioinformatics ( Accession No. Xp Essay
Genetic Dna And Bioinformatics ( Accession No. Xp Essay
 
QS
QSQS
QS
 
CHEM3204_PRAC_Manual_2016
CHEM3204_PRAC_Manual_2016CHEM3204_PRAC_Manual_2016
CHEM3204_PRAC_Manual_2016
 
ISSCR-2014-poster
ISSCR-2014-posterISSCR-2014-poster
ISSCR-2014-poster
 
Research Symposium Poster Draft
Research Symposium Poster DraftResearch Symposium Poster Draft
Research Symposium Poster Draft
 
Seminario biología molecular María de los Ángeles Montoya
Seminario biología molecular María de los Ángeles MontoyaSeminario biología molecular María de los Ángeles Montoya
Seminario biología molecular María de los Ángeles Montoya
 
PhD_pages_Linkdin_English
PhD_pages_Linkdin_EnglishPhD_pages_Linkdin_English
PhD_pages_Linkdin_English
 
Grindberg - PNAS
Grindberg - PNASGrindberg - PNAS
Grindberg - PNAS
 
A panel of recombinant monoclonal antibodies against zebrafish
A panel of recombinant monoclonal antibodies against zebrafishA panel of recombinant monoclonal antibodies against zebrafish
A panel of recombinant monoclonal antibodies against zebrafish
 

Mehr von Prasit Chanarat

Mehr von Prasit Chanarat (20)

เหรียญดุษฎีมาลา เข็มศิลปวิทยา
เหรียญดุษฎีมาลา เข็มศิลปวิทยาเหรียญดุษฎีมาลา เข็มศิลปวิทยา
เหรียญดุษฎีมาลา เข็มศิลปวิทยา
 
Watchara
WatcharaWatchara
Watchara
 
Blood agar
Blood agarBlood agar
Blood agar
 
Polycythemia
PolycythemiaPolycythemia
Polycythemia
 
Hemopoisis
HemopoisisHemopoisis
Hemopoisis
 
Hemopoiesis[med]
Hemopoiesis[med]Hemopoiesis[med]
Hemopoiesis[med]
 
Mt research
Mt researchMt research
Mt research
 
บทเรียน ทำ สปาเกตตี
บทเรียน ทำ สปาเกตตีบทเรียน ทำ สปาเกตตี
บทเรียน ทำ สปาเกตตี
 
โครงการส่งเสริมสุขภาพชุมชน
โครงการส่งเสริมสุขภาพชุมชนโครงการส่งเสริมสุขภาพชุมชน
โครงการส่งเสริมสุขภาพชุมชน
 
Naturally acquired plasmodium knowlesi malaria in human, thailand[1]
Naturally acquired plasmodium knowlesi malaria in human, thailand[1]Naturally acquired plasmodium knowlesi malaria in human, thailand[1]
Naturally acquired plasmodium knowlesi malaria in human, thailand[1]
 
Goa
GoaGoa
Goa
 
neutro
neutroneutro
neutro
 
idf
idfidf
idf
 
typhoid
typhoidtyphoid
typhoid
 
rprotein1
rprotein1rprotein1
rprotein1
 
rprotein
rproteinrprotein
rprotein
 
recombinant_protein_handbook
recombinant_protein_handbookrecombinant_protein_handbook
recombinant_protein_handbook
 
rprotein
rproteinrprotein
rprotein
 
hemato in systemic diseases
hemato in systemic diseaseshemato in systemic diseases
hemato in systemic diseases
 
Hemato in systemic diseases
Hemato in systemic diseasesHemato in systemic diseases
Hemato in systemic diseases
 

rprotein3

  • 1. Protein Engineering vol.15 no.4 pp.337–345, 2002 Engineering a novel secretion signal for cross-host recombinant protein expression Nguan Soon Tan1,2, Bow Ho3 and Jeak Ling Ding1,4 tailoring to meet the stringent requirements for each protein 1Department 3Department product to ensure correct folding, activity and desired yield. of Biological Sciences and of Microbiology, National University of Singapore, Singapore 117543 Furthermore, the flexibility of a common secretion signal 4To sequence with which to secrete a wide variety of heterologous whom correspondence should be addressed at: Department of Biological Sciences, National University of Singapore, 10 Kent Ridge Crescent, fusion proteins from various hosts into the extracellular medium Science Drive 4, Singapore 117543. is not available. E-mail: dbsdjl@nus.edu.sg Protein secretion is one of the most important issues of 2Present address: Institut de Biologie Animale, Batiment de Biologie, protein expression in fundamental processes of living cells. Universite de Lausanne, CH-1015, Lausanne, Switzerland Successful protein secretion requires effective translocation of Protein secretion is conferred by a hydrophobic secretion the protein across the endoplasmic recticulum or plasma signal usually located at the N-terminal of the polypeptide. membrane. Proteins destined for secretion are targeted to the We report here, the identification of a novel secretion signal membrane via their respective secretion signals that are usually (SS) that is capable of directing the secretion of recombinant located at the N-terminal of nascent polypeptides. These signals proteins from both prokaryotes and eukaryotes. Secretion display very little primary sequence conservation. However, of fusion reporter proteins was demonstrated in Escherichia they all possess three general domains: an N-terminal region coli, Saccharomyces cerevisiae and six different eukaryotic that varies widely in length, but typically, contain amino acids cells. Estrogen-inducibility and secretion of fusion reporter which contribute a net positive charge to this domain; a central protein was demonstrated in six common eukaryotic cell hydrophobic region made up of a block of seven to 16 lines. The rate of protein secretion is rapid and its expres- hydrophobic amino acids; and a C-terminal region that includes sion profile closely reflects its intracellular concentration the signal cleavage site (Nothwehr and Gordon, 1990; Pines of mRNA. In bacteria and yeast, protein secretion directed and Inouye, 1999). Since the principles of protein transloca- tion mechanism are evolutionarily conserved (Schatz and by SS is dependent on the growth culture condition and Dobberstein, 1996), it is conceivable that there exists a secretion rate of induction. This secretion signal allows a flexible signal that is operational in both prokaryote and eukaryote, strategy for the production and secretion of recombinant viz, cross-host. proteins in numerous hosts, and to conveniently and rapidly Our previous efforts to express and secrete the limulus study protein expression. Factor C, a highly complex serine protease, in Escherichia Keywords: broad hosts/protein expression/secretion signal coli, Pichia pastoris and COS cells using its native hydrophobic signal, Saccharomyces cerevisiae α-mating factor or Kluyveromyces lactis killer toxin secretion signal were unsuc- Introduction cessful (Roopashree et al., 1995, 1996; and unpublished data). With innovative genomics technology, genes are being disco- Surprisingly, the secretion of the similar construct was achieved vered faster than their functions can be characterized. As we by a novel 15-residue hydrophobic secretion signal (SS) in enter the era of proteomics, the ability to rapidly produce large Drosophila S2 cells (Tan et al., 2000a). Furthermore, varying numbers of proteins in a parallel manner becomes increasingly the genes in the fusion or the tags, did not affect the high- important. Determining their functions requires numerous level secretion and cleavage at the correct site (Tan et al., biophysical (e.g. crystallization, NMR, MS) and functional 2000a,b). Despite the origin of this signal, 80% of the studies (e.g. protein–protein interactions), each of which uses recombinant proteins expressed by the heterologous insect host a different expression vector. Hindrances to these analyses were localized in the extracellular medium. In this study, include the arduous task of subcloning, problems with reading we demonstrate the versatility and functionality of SS for frame and Kozak sequence, as well as the downstream recombinant protein expression and secretion in cross-hosts purification protocols. A versatile system for transferring DNA [E.coli, S.cerevisiae, higher eukaryotic cells—African green fragments between vectors using the Cre-lox recombinase monkey kidney cells (COS-1), Chinese hamster ovary cells technology has been recently developed (Liu et al., 1998). In (CHO-B), fibroblast cells derived from Swiss mouse embryo addition, the Sindbis expression system enables the rapid, (NIH/3T3), human cervical adenocarcinoma cells (HeLa), carp high-level expression of heterologous proteins in a variety of epithelial cells (EPC) and chinook salmon embryonic cells eukaryotic cell lines derived from mammalian, avian and insect (CHSE)]. The expression and secretion of the recombinant hosts (Xiong et al., 1989). Recombinant proteins synthesized proteins were performed using either a constitutive or an in heterologous hosts may accumulate in one of three ‘compart- inducible promoter. In addition, we compared the secretion ments’: the cytoplasm, the periplasm or the extracellular rate of reporter protein directed by SS and human secreted medium. Many overexpressed proteins from various origins alkaline phosphatase (SEAP) signal, and assessed the efficiency have been purified from each of these locations. Whenever of secretion in different yeast media. This paper illustrates the possible, secretion is the preferred strategy since it permits engineering of SS to aid the production of secreted recombinant easy and efficient purification from the extracellular medium. protein for easier analysis. To the best of our knowledge, this However, to date, each expression system needs specific study documents the only known cross-host secretion signal. © Oxford University Press 337
  • 2. N.S.Tan, B.Ho and J.L.Ding 338
  • 3. 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 the manufacturer. 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. 339
  • 4. 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 SEAP. 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 340
  • 5. 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 341
  • 6. 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 bacteria 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. 342
  • 7. 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 343
  • 8. 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 SS-β-galactosidase) and eukaryotic proteins (EGFP), regardless References of protein size. Moreover, the rate of secretion of heterologous Allet,B., Bernard,A.R., Hochmann,A., Rohrbach,E., Graber,P., Magnenat,E., proteins is at least 3-fold faster than the SEAP native signal Mazzei,G.J. and Bernasconi,L. (1997) Protein Expr. Purif., 9, 61–68. Asakura,A., Minami,M. and Ota,Y. (1995) Biosci. Biotechnol. Biochem., 59, sequence. Taken together, SS is the only signal sequence 1976–1978. known to date that is functional in all four common expression Baldari,C., Murray,J.A., Ghiara,P., Cesareni,G. and Galeotti,C.L. (1987) EMBO hosts (Table I). J., 6, 229–234. What makes SS such an efficient universal secretion signal? Blight,M.A. and Holland,I.B. (1994) Trends Biotechnol., 12,450–455. Brake,A.J., Merryweather,J.P., Coit,D.G., Heberlein,U.A., Masiarz,F.R., SS is capable of cross-host secretion for several reasons. First, Mullenbach,G.T., Urdea,M.S., Valenzuela,P. and Barr,P.J. (1984) Proc. Natl it has the three domains typified in all secretion signals and Acad. Sci. USA, 81, 4642–4646. the presence of small amino acid residues at position –1 and – Chen,D.C., Yang,B.C. and Kuo,T.T. (1992) Curr. Genet., 21, 83–84. 3 of the cleavage site (Jain et al., 1994). Secondly, the charge Chervaux,C., Sauvonnet,N., Le Clainche,A., Kenny,B., Hung,A.L., Broome- to hydrophobicity ratio between the N-terminal domain and Smith,J.K. and Holland,I.B. (1995) Mol. Gen. Genet., 249, 237–245. Cohenford,M.A., Abraham,J. and Medeiros,A.A. (1988) Anal. Biochem., 168, hydrophobic core, which is important for directing the protein 252–258. to the membrane (Rusch et al., 1994; Izard et al., 1996), Coloma,M.J., Hastings,A., Wims,L.A. and Morrison,S.L. (1992) J. Immunol. represents a compromise of the requirements needed by both Methods, 152, 89–104. the prokaryotic and eukaryotic hosts. Lastly, while many Farrell,P.J., Behie,L.A. and Iatrou,K. (1999) Biotechnol. Bioeng., 64, 426–433. Golden,A., Austen,D.A., van Schravendijk,M.R., Sullivan,B.J., Kawasaki,E.S. currently available secretion expression vectors also satisfy and Osburne,M.S. (1998) Protein Expr. Purif., 14, 8–12. the first criteria, few possess the optimal ratio with respect to Gray,G.L., Baldridge,J.S., McKeown,K.S., Heyneker,H.L. and Chang,C.N. criteria two, and none of them address the issue of sequences (1985) Gene, 39, 247–254. beyond the cleavage site, i.e. C-terminal, necessary for effective Hitzeman,R.A., Chen,C.Y., Hagie,F.E., Lugovoy,J.M. and Singh,A. (1984) In and homogenous cleavage and thus secretion. It is conceivable ed. Arthur P.Bollon Recombinant DNA Products: Insulin, Interferon and Growth Hormone. CRC Press, Boca Raton, FL. that the C-terminal sequences are able to tolerate more degener- Itakura,K., Hirose,T., Crea,R., Riggs,A.D., Heyneker,H.L., Bolivar,F. and acy and hence the apparent redundancy to highlight this Boyer,H.W. (1977) Science, 198, 1056–1063. criteria. Effective cross-host secretion, in our case, requires Izard,J.W., Rusch,S.L. and Kendall,D.A. (1996) J. Biol. Chem., 271, 21579– this neglected criterion to be resolved. Initial attempts to 21582. Jain,R.G., Rusch,S.L. and Kendall,D.A. (1994) J. Biol. Chem., 269, 16305– reduce and/or remove the post-cleavage remnant residues 16310. resulted in non-secreted recombinant protein (unpublished Kirkpatrick,R.B., Ganguly,S., Angelichio,M., Griego,S., Shatzman,A., data). This compromise of post-cleavage six residues in the Silverman,C. and Rosenberg,M. (1995) J. Biol. Chem., 270, 19800–19805. recombinant proteins is highly unlikely to alter the recombinant Kjeldsen,T. (2000) Appl. Microbiol. Biotechnol., 54, 277–286. protein functions. Comparatively, the post-cleavage remnant Laforet,G.A and Kendall,D.A. (1991) J. Biol. Chem., 266, 1326–1334. Laroche,Y., Storme,V., De Meutter,J., Messens,J. and Lauwereys,M. (1994) residues of SS are significantly smaller than GST or GFP and Biotechnology, 12, 1119–1124. possess lesser charge than the 6 histidine tag. While this work Lasky,L.A., Groopman,J.E., Fennie,C.W., Benz,P.M., Capon,D.J., clearly documents SS as a cross-host secretion signal, the Dowbenko,D.J., Nakamura,G.R., Nunes,W.M., Renz,M.E. and Berman,P.W. functionality of the secreted recombinant protein produced by (1986) Science, 233, 290–212. Laukkanen,M.L., Oker-Blom,C. and Keinanen,K. (1996) Biochem. Biophys. any particular host will also strongly depend on other factors Res. Commun., 226, 755–761. such as post-translational modifications and intrinsic properties Liu,Q., Li,M.Z., Leibham,D., Cortez,D. and Elledge,S.J. (1998) Curr. Biol., of the protein to be expressed. 8, 1300–1309. 344
  • 9. Secretion signal for protein expression Nothwehr,S.F. and Gordon,J.I. (1990) BioEssays, 12, 479–484. Pines,O. and Inouye,M. (1999) Mol. Biotechnol., 12, 25–34. Rapoza,M.P. and Webster,R.E. (1993) J. Bacteriol., 175, 1856–1859. Roopashree,S.D., Chai,C., Ho,B. and Ding,J.L. (1995) Biochem. Mol. Biol. Int., 35, 841–849. Roopashree,S.D., Ho,B. and Ding,J.L. (1996) Mol. Marine Biol. Biotechnol., 5, 334–343. Rusch,S.L., Chen,H., Izard,J.W. and Kendall,D.A. (1994) J. Cell Biochem., 55, 209–217. Schatz,G. and Dobberstein,B. (1996) Science, 271, 1519–1526. Sleep,D., Belfield,G.P. and Goodey,A.R. (1990) Biotechnology, 8, 42–46. Sykes,R.B. and Mathew,M. (1976) J. Antimicrob. Chemother., 2, 115–157. Takahara,M., Sagai,H., Inouye,S. and Inouye,M. (1988) Bio/Technology, 6, 195–198. Tan,N.S., Frecer,V., Lam,T.J. and Ding,J.L. (1999) Biochim. Biophys. Acta, 1452, 103–120. Tan,N.S., Ho,B. and Ding,J.L. (2000a) FASEB J., 14, 859–870. Tan,N.S., Ng,P.M.L., Yau,Y.H., Chong,P.K.W., Ho,B. and Ding,J.L. (2000b) FASEB J., 14, 1801–1813. Tessier,D.C., Thomas,D.Y., Khouri,H.E., Laliberte,F. and Vernet,T. (1991) Gene, 98, 177–183. Uhlen,M. and Abrahmsen,L. (1989) Biochem. Soc. Trans., 17, 340–341. Wang,J., Ho,B. and Ding,J.L. (2001) Biotechnol. Lett., 23, 71–76. Waxman,D.J. and Strominger,J.L. (1983) Annu. Rev. Biochem., 52, 825–869. Xiong,C., Levis,R., Shen,P., Schlesinger,S., Rice,C.M. and Huang,H.V. (1989) Science, 243, 1188–1191. Received May 25, 2001; revised December 18, 2001; accepted January 4, 2002 345