Protein Engineering, Vol. 15, No. 4, 337-345,
April 2002
© 2002 Oxford University Press
Engineering a novel secretion signal for cross-host recombinant protein expression
1 Department of Biological Sciences and 3 Department of Microbiology, National University of Singapore, Singapore 117543
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Abstract |
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Protein secretion is conferred by a hydrophobic secretion signal usually located at the N-terminal of the polypeptide. We report here, the identification of a novel secretion signal (SS) that is capable of directing the secretion of recombinant proteins from both prokaryotes and eukaryotes. Secretion of fusion reporter proteins was demonstrated in Escherichia coli, Saccharomyces cerevisiae and six different eukaryotic cells. Estrogen-inducibility and secretion of fusion reporter protein was demonstrated in six common eukaryotic cell lines. The rate of protein secretion is rapid and its expression profile closely reflects its intracellular concentration of mRNA. In bacteria and yeast, protein secretion directed by SS is dependent on the growth culture condition and rate of induction. This secretion signal allows a flexible strategy for the production and secretion of recombinant proteins in numerous hosts, and to conveniently and rapidly study protein expression.
Keywords: broad hosts/protein expression/secretion signal
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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With innovative genomics technology, genes are being discovered faster than their functions can be characterized. As we enter the era of proteomics, the ability to rapidly produce large numbers of proteins in a parallel manner becomes increasingly important. Determining their functions requires numerous biophysical (e.g. crystallization, NMR, MS) and functional studies (e.g. protein–protein interactions), each of which uses a different expression vector. Hindrances to these analyses include the arduous task of subcloning, problems with reading frame and Kozak sequence, as well as the downstream purification protocols. A versatile system for transferring DNA fragments between vectors using the Cre-lox recombinase technology has been recently developed (Liu et al., 1998
). In addition, the Sindbis expression system enables the rapid, high-level expression of heterologous proteins in a variety of eukaryotic cell lines derived from mammalian, avian and insect hosts (Xiong et al., 1989). Recombinant proteins synthesized in heterologous hosts may accumulate in one of three `compartments': the cytoplasm, the periplasm or the extracellular medium. Many overexpressed proteins from various origins have been purified from each of these locations. Whenever possible, secretion is the preferred strategy since it permits easy and efficient purification from the extracellular medium. However, to date, each expression system needs specific tailoring to meet the stringent requirements for each protein product to ensure correct folding, activity and desired yield. Furthermore, the flexibility of a common secretion signal sequence with which to secrete a wide variety of heterologous fusion proteins from various hosts into the extracellular medium is not available.
Protein secretion is one of the most important issues of protein expression in fundamental processes of living cells. Successful protein secretion requires effective translocation of the protein across the endoplasmic recticulum or plasma membrane. Proteins destined for secretion are targeted to the membrane via their respective secretion signals that are usually located at the N-terminal of nascent polypeptides. These signals display very little primary sequence conservation. However, they all possess three general domains: an N-terminal region that varies widely in length, but typically, contain amino acids which contribute a net positive charge to this domain; a central hydrophobic region made up of a block of seven to 16 hydrophobic amino acids; and a C-terminal region that includes the signal cleavage site (Nothwehr and Gordon, 1990; Pines and Inouye, 1999). Since the principles of protein translocation mechanism are evolutionarily conserved (Schatz and Dobberstein, 1996), it is conceivable that there exists a secretion signal that is operational in both prokaryote and eukaryote, viz, cross-host.
Our previous efforts to express and secrete the limulus Factor C, a highly complex serine protease, in Escherichia coli, Pichia pastoris and COS cells using its native hydrophobic signal, Saccharomyces cerevisiae 
-mating factor or Kluyveromyces lactis killer toxin secretion signal were unsuccessful (Roopashree et al., 1995, 1996; and unpublished data). Surprisingly, the secretion of the similar construct was achieved by a novel 15-residue hydrophobic secretion signal (SS) in Drosophila S2 cells (Tan et al., 2000a). Furthermore, varying the genes in the fusion or the tags, did not affect the high-level secretion and cleavage at the correct site (Tan et al., 2000a,b). Despite the origin of this signal, >80% of the recombinant proteins expressed by the heterologous insect host were localized in the extracellular medium. In this study, we demonstrate the versatility and functionality of SS for recombinant protein expression and secretion in cross-hosts [E.coli, S.cerevisiae, higher eukaryotic cells—African green monkey kidney cells (COS-1), Chinese hamster ovary cells (CHO-B), fibroblast cells derived from Swiss mouse embryo (NIH/3T3), human cervical adenocarcinoma cells (HeLa), carp epithelial cells (EPC) and chinook salmon embryonic cells (CHSE)]. The expression and secretion of the recombinant proteins were performed using either a constitutive or an inducible promoter. In addition, we compared the secretion rate of reporter protein directed by SS and human secreted alkaline phosphatase (SEAP) signal, and assessed the efficiency of secretion in different yeast media. This paper illustrates the engineering of SS to aid the production of secreted recombinant protein for easier analysis. To the best of our knowledge, this study documents the only known cross-host secretion signal.
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Materials and methods |
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Construction of secretory CAT and ß-galactosidase expression vectors
The isolation and initial cloning of SS into pEGFP-N1, to yield pSSEGFP was described in Tan et al. (Tan et al., 2000a). Detailed sequences and cloning strategies of secretory chloramphenicol acetyltransferase (SSCAT) and ß-galactosidase (SS-Gal) can be obtained from the corresponding author. The vector maps of various constructs are illustrated in Figure 1.
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Cell culture and transfections
COS-1, NIH/3T3, HeLa and CHO-B cells were maintained in DMEM while EPC and CHSE were cultured in MEM. All media were phenol-red free and supplemented with 10% charcoal/dextran-treated fetal bovine serum. Cells were transfected with 1 µg of SS-fusion construct:control vector in a ratio of 8:2, by lipofectamine (Gibco BRL) as described by the manufacturer.
For estrogen-induction experiment, cells were co-transfected with ERU-psp-SSCAT, pSGcER (chicken estrogen receptor) and pSEAP-Control as described in Tan et al. (Tan et al., 1999). For studies on the rate of secretion, better comparison between the CAT and ß-galactosidase ELISA were achieved by adapting to fluorescence assays using the DIG Fluorescence Detection ELISA (Boehringer Mannheim). SEAP was determined fluorimetrically (LS-50B, Perkin Elmer) at Ex360nm and Em449nm. SSCAT and SS-Gal were detected at Ex440nm and Em550nm.
Expression of SSCAT in S.cerevisiae
The construct pYEX-SSCAT was transformed into S.cerevisiae DY150 (Chen et al., 1992). The transformants were selected on synthetic minimal medium (MM) agar containing all the required supplements except uracil. For expression analysis, a 100 ml YEPD medium (2% yeast extract, 1% mycopeptone, 2% D-glucose, 5x HTA: 100 mg each of histidine, tryptophan and adenine, pH 5.0) was inoculated with a single clone and grown for 16 h at 30°C. Subsequently, 50 ml of the yeast cultures were grown independently for 72 h in 2x1 l baffled flasks containing either 200 ml of YEPD or MM. At indicated time intervals, 2 ml aliquots of culture were centrifuged to obtain yeast pellet and culture supernatant. The yeast cells were lysed with glass beads (Roopashree et al., 1996), whereas the culture medium was collected and frozen without any pre-treatment. The pH of the culture was adjusted to pH 5.0 using 1 M potassium phosphate buffer (pH 8.0). SSCAT was measured as described above.
Arabinose-induced expression of modified SS-ß-lactamase in bacteria
Transformants of E.coli LM194 with pBADSSblactKana were selected for by plating on LB agar containing 50 µg/ml kanamycin. For the ampicillin plate assay, LM194 competent bacteria were transformed and plated on ampicillin LB agar plates containing 0.2% glucose or at increasing dosage of arabinose. The expression and secretion of functional SS-ß-lactamase was visualized as colony formation.
For liquid assay, 5 ml of RM medium (1x M9 salts, 2% casamino acids, 0.2% D-glucose, 1 mM MgCl2, 50 µg/ml kanamycin) was inoculated with either a single recombinant or untransformed LM194 colony. The induction procedure was as described by the manufacturer (Invitrogen). Prior to induction, a 1 ml aliquot of culture was removed, processed and designated the zero time point. The medium was clarified off bacteria by centrifugation and sterile filtered using a 0.22 µm membrane. The periplasmic space fraction was isolated from the cell lysate (Laforet and Kendall, 1991). Both the medium and periplasmic fraction were assayed for ß-lactamase activity (Cohenford et al., 1988).
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Results |
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SS directs the secretion of reporter protein into culture medium
To investigate whether SS can direct the secretion of common reporter proteins from various eukaryotic hosts, fusion constructs of SS with CAT and ß-galactosidase driven by constitutive CMV or SV40 promoter, were transfected into a variety of cell lines, namely COS-1, NIH/3T3, CHO-B, EPC, HeLa and CHSE. As illustrated in Figure 2, SSCAT, ssEGFP and SS-Gal were effectively secreted and accumulated in the culture medium of all the cell lines tested, although the amount of SS-fusion protein produced varied. Despite being diluted in the culture medium, the secreted recombinant proteins were detectable within 24 h, indicating high level expression.
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Rapid secretion rate of SSCAT and SS-Gal compared to SEAP
The amount of SS-fusion protein secreted at various time intervals was determined using a standard curve generated from the positive control provided by the kits. The rate of secretion was determined by the gradient of the best-fit line when the amount of secreted protein was plotted against time. The mean rates of secretion of SSCAT and SS-Gal were 15.8 fg/ml SSCAT/ng ß-galactosidase/min ± 0.12 fg/ml/min and 12.1 fg/ml SS-Gal/ng SEAP/min ± 0.09 fg/ml/min, respectively (Figure 3). In comparison, SEAP was secreted at a rate of 4.76 fg/ml SEAP/ng ß-galactosidase/min ± 0.06 fg/ml/min. The rate of SSCAT and SS-Gal secretion was almost 3-fold higher than SEAP. Thus, this indicates that there is a rapid post-translational processing of the SS.
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Inducible expression of SSCAT protein correlated with its mRNA level
The expression and secretion of SS-fusion proteins, in particular SSCAT, were also examined using an inducible promoter. The estrogen-induced expression and secretion of SSCAT was observed in all the cell lines tested, although the amount of SSCAT produced varied. Uninduced COS-1 cells exhibited only marginal increase in SSCAT over a period of 24 h. For induced COS-1 cells, the increase in SSCAT can be detected as early as 2 h, reaching a peak of 7-fold increase at 12 h post-induction (Figure 4a). Estrogen-induced expression of SSCAT can also be observed in other vertebrate cell lines, namely NIH/3T3, CHO-B and EPC cells (Figure 4b).
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To verify that the level of secreted SSCAT in the culture 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 previously observed rapid secretion of SS-fusion proteins driven by constitutive promoters is not due to the strength of the promoter, but rather, the properties of SS as a secretion signal. Thus far, we have demonstrated that SS is functional in several common higher eukaryotic hosts, both mammalian and non-mammalian. In addition, under similar experimental conditions, a higher level of SS-fusion proteins was detected in the extracellular medium as compared to SEAP. The novel SS can direct secretion of recombinant proteins in yeast
Although, recombinant protein expression in yeast has its limitations, it is still a favorable choice because it can be cultivated readily in large-scale fermentation, with an advantage of releasing relatively little extraneous protein material into the medium and post-translational modifications of proteins. To further examine the versatility of SS, the secretion of SS-fusion protein, SSCAT, driven by constitutive PGK promoter was studied in two independent S.cerevsiae transformants cultured in two different media.
The SSCAT expression profile was monitored over 72 h in yeast grown in YEPD (rich medium) and MM (minimal medium). After 24–48 h of culture, the yeast transformants grown in MM secreted significantly less SSCAT in the medium. It is unlikely that the overall SSCAT expression was reduced in MM-cultured yeast since comparable SSCAT protein was observed in the yeast lysate of both MM and YEPD cultures. Interestingly, the decrease corresponds to a drop in the pH of MM. Adjusting the pH back to 5, resulted in a tremendous increase in secreted SSCAT. This effect is less pronounced in the rich YEPD medium, probably because it supports high-density growth and has higher buffering capacity. The amount of SSCAT detected in both types of culture media was comparable at 72 h (Figure 5).
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It is noteworthy that although the amount of SSCAT in the medium is
50% that of yeast lysate, this is likely an 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-ß-lactamase in bacteria
Ampicillin which belongs to the ß-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 ß-lactamase, and is secreted into the periplasmic space of the bacterium, where it catalyzes hydrolysis of the ß-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 ß-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 ß-lactamase, SS-ß-lactamase, where its native secretion signal was replaced by SS in a construct, pBADSSblactKana. The expression of the SS-ß-lactamase came under the control of an inducible arabinose-responsive promoter. As illustrated in Figure 6a, no colonies were seen in the absence or presence of 0.0002% arabinose or 0.2% glucose alone, whereas dose-dependent arabinose-induced (0.002–0.2%) expression and secretion of SS-ß-lactamase permitted the bacterial transformants to survive on ampicillin LB agar plates. Interestingly, the colony size appeared distinctively smaller with increasing levels of arabinose.
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To further examine the efficacy of SS directed ß-lactamase secretion, we decided to measure SS-ß-lactamase activities via a liquid assay. The pBADSSblactKana clone grown in RM medium with 0.2% glucose (i.e. no induction) exhibited a similar growth profile as the control LM194 host bacteria (data not shown). Concomitant with the plate assay, no SS-ß-lactamase activity can be detected in the culture medium and periplasmic space (Figure 6b and c
) of uninduced transformants. The addition of arabinose resulted in expression and accumulation of SS-ß-lactamase in the culture medium and periplasmic space. Even more surprising is that the highest level of enzyme was detected when 0.0002% arabinose was used (Figure 6c). This apparent conflict was due to the growth-inhibitory effect on the bacteria when induced at a high concentration of arabinose (data not shown). The dose-dependent expression profile of SS-ß-lactamase, however, was not observed after 4 h. Similar results were obtained using the TOP10 strain of E.coli, although the overall protein expression level decreased by 20% (data not shown). |
Discussion |
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The fundamental basis for the search of a cross-host secretion signal really lies in the efficacy between heterologous versus homologous secretion signals. Heterologous secretion signals refer to the use of this signal for the secretion of heterologous gene products, as well as from a different host from which the signal sequence was derived. In contrast, homologous secretion signals refer to the secretion of its natural gene product from the same host species. Certainly, a cross-host secretion signal will have to satisfy three other important criteria: (i) this signal must confer secretion to gene products of different origins (prokaryotic or eukaryotic); (ii) the functionality of this signal must extend beyond its original host; (iii) the expression and secretion of the gene products must be of appreciable quantity and functional. Currently, no single secretion signal has been demonstrated to be effective in both prokaryotic and eukaryotic host expression systems. The currently available secretion signals have exhibited limited functionality and/or non-compatibility for cross-host recombinant protein expression (Table I
). Therefore, availability of a common broad-host secretion signal is highly desirable. The major objective of this study was to evaluate the efficiency of SS in directing cross-host expression and secretion of foreign proteins. Consequently, SS was subcloned upstream of three reporter protein genes—EGFP, CAT and ß-galactosidase. These three proteins were chosen because of their different size and origin (prokaryotic versus eukaryotic).
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Based on strict definition, no functional heterologous secretion signal was reported for bacterial use. Although the expression and secretion of numerous heterologous genes, such as human superoxide dismutase (Takahara et al., 1988
), have been successful in bacteria, most if not all bacterial expression utilized secretion signals of prokaryotic origin (Table I). Perhaps, the closest example was that of human growth hormone (hGH). Gray et al. (Gray et al., 1985) compared the efficiency of export of hGH directed by either its own signal sequence or the E.coli Pho A signal sequence. Results indicated that the secretions are comparable with 72% of the hGH localized in the periplasm. However, the efficacy of the hGH signal in directing the secretion of heterologous proteins in bacteria has not been reported. In comparison, the potential of SS to direct secretion of proteins in E.coli was evaluated by the secretion of a modified SS-ß-lactamase. Via plate and liquid assays, we showed that the secretion is rapid and at least 50% of the protein is detectable in the extracellular medium upon induction. However, high doses of the inducer, arabinose, led to lower secreted product. Overloading the export machinery may result from inefficient secretion of a foreign protein because the protein is expressed at levels that simply exceed cellular capacity. This is the first report of a functional heterologous signal sequence in bacteria that permits appreciable yield of secreted recombinant protein.
The first heterologous secretion signal for yeast was the human serum albumin (hHSA). This human secretion signal works well in yeast, producing 50% of the hHSA in yeast fermentation media (Sleep et al., 1990). This signal results not only in the hHSA secretion but also the secretion and desired processing of other heterologous genes, such as human immunodeficiency virus (HIV) gp120 (Lasky et al., 1986) and somatostatin (Itakura et al., 1977). Again, the functionality of hHSA signal in bacteria was not reported. Interestingly, expression of hGH in yeast results in properly processed hGH in yeast media, suggesting that the signal recognition is not flawed. However, only 10% of the expressed protein is secreted whereas 90% of hGH remains cell-associated and retains the entire signal sequence (Hitzeman et al., 1984). In comparison, we used SS to direct the secretion of a prokaryotic protein, SSCAT. As shown in Figure 5, at least 50% of the protein was secreted into the yeast media. Unlike, the hHSA signal sequence, SS is applicable in bacteria. It is worth noting that in rapidly growing expression hosts, such as that of E.coli and S.cerevisiae, the rate of secretion is greatly influenced by their growth conditions. Consequently, for optimal secretion of recombinant protein via SS, in rapidly growing expression hosts, a compromise must be struck between growth condition and concentration of the inducer, in order to regulate the rate of recombinant protein production and its secretion.
Many secreted eukaryotic proteins are efficiently processed in mammalian expression host via their native signal sequences. Hence, a more comprehensive study was done with SS. The SS is able to direct secretion of both prokaryotic (SSCAT and SS-ß-galactosidase) and eukaryotic proteins (EGFP), regardless of protein size. Moreover, the rate of secretion of heterologous proteins is at least 3-fold faster than the SEAP native signal sequence. Taken together, SS is the only signal sequence known to date that is functional in all four common expression hosts (Table I).
What makes SS such an efficient universal secretion signal? SS is capable of cross-host secretion for several reasons. First, it has the three domains typified in all secretion signals and the presence of small amino acid residues at position –1 and –3 of the cleavage site (Jain et al., 1994). Secondly, the charge to hydrophobicity ratio between the N-terminal domain and hydrophobic core, which is important for directing the protein to the membrane (Rusch et al., 1994; Izard et al., 1996), represents a compromise of the requirements needed by both the prokaryotic and eukaryotic hosts. Lastly, while many currently available secretion expression vectors also satisfy the first criteria, few possess the optimal ratio with respect to criteria two, and none of them address the issue of sequences beyond the cleavage site, i.e. C-terminal, necessary for effective and homogenous cleavage and thus secretion. It is conceivable that the C-terminal sequences are able to tolerate more degeneracy and hence the apparent redundancy to highlight this criteria. Effective cross-host secretion, in our case, requires this neglected criterion to be resolved. Initial attempts to reduce and/or remove the post-cleavage remnant residues resulted in non-secreted recombinant protein (unpublished data). This compromise of post-cleavage six residues in the recombinant proteins is highly unlikely to alter the recombinant protein functions. Comparatively, the post-cleavage remnant residues of SS are significantly smaller than GST or GFP and possess lesser charge than the 6 histidine tag. While this work clearly documents SS as a cross-host secretion signal, the functionality of the secreted recombinant protein produced by any particular host will also strongly depend on other factors such as post-translational modifications and intrinsic properties of the protein to be expressed.
This study reports the identification and development of a cross-host secretion signal. Its ability to direct recombinant protein secretion was evaluated with SS-fusion reporter proteins in various hosts—higher eukaryote, yeast and bacteria. We envision that fusion of the SS to recombinant genes will prove to be a valuable tool for efficient protein secretion in a broad heterologous host expression system. This secretion signal can be incorporated into the `donor vector' of various multi-vector cloning systems, such as GatewayTM (Gibco BRL) and EchoTM Cloning (Invitrogen), which can then be transferred into various host expression vectors for expression and secretion of recombinant proteins. This secretion signal can also be incorporated into various reporter assay systems for rapid, and minimal set-up reporter gene analyses. While an exhaustive screen of all proteins is beyond the scope of this study, during the process of preparing this paper, the SS has been further evaluated by other researchers and was proven to yield varied success in the secretion of other recombinant proteins, for example, in Drosophila S2 cells, Sf9 cells (Wang et al., 2001) and E.coli (unpublished data).
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Notes |
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4 To whom correspondence should be addressed at: Department of Biological Sciences, National University of Singapore, 10 Kent Ridge Crescent, Science Drive 4, Singapore 117543. E-mail: dbsdjl{at}nus.edu.sg
2 Present address: Institut de Biologie Animale, Batiment de Biologie, Universite de Lausanne, CH-1015, Lausanne, Switzerland
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Acknowledgments |
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We thank Professor W.Wahli for Xenopus Vtg B1 ERU, and Professor P.Chambon for pSG cER. This work was funded by NUS Grant RP3999900/A and NSTB Grant LS/99/004.
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Received May 25, 2001; revised December 18, 2001; accepted January 4, 2002.
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