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  • 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. 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