PEDS Advance Access originally published online on May 19, 2006
Protein Engineering Design and Selection 2006 19(7):337-343; doi:10.1093/protein/gzl017
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Extracellular secretion of Escherichia coli alkaline phosphatase with a C-terminal tag by type I secretion system: purification and biochemical characterization
1 Department of Material and Life Science, Graduate School of Engineering, Osaka University 2-1 Yamadaoka, Suita, Osaka 565-0871 2 Discovery Research Laboratory, Tanabe Seiyaku Co., Ltd. 2-50, Kawagishi-2-chome, Toda, Saitama 335-8505 3 Presto, JST 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
4To whom correspondence should be addressed. E-mail: kanaya{at}mls.eng.osaka-u.ac.jp
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Abstract |
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Type I secretion system (TISS) of Gram-negative bacteria permits proteins to be secreted directly from the cytoplasm to the external medium by a single, energy-coupled step. To examine whether this system can be used as an extracellular production system of recombinant proteins, Escherichia coli alkaline phosphatase (AP) was fused to a C-terminal region of Pseudomonas sp. MIS38 lipase (PML) and examined for secretion using the E.coli cells carrying the heterologous TISS. PML is one of the passenger proteins of TISS and contains 12 repetitive sequences and a secretion signal at the C-terminal region. The fusion protein was efficiently secreted to the extracellular medium, while AP was not secreted at all, indicating that the secretion of AP is promoted by a secretion signal of PML. The repetitive sequences were not so important for secretion of the fusion protein, because the secretion level of the fusion protein containing entire repeats (
10 mg/l culture) was only 2-fold higher than that of the fusion protein without repeats. The fusion protein purified from the culture supernatant existed as a homodimer, like AP, and was indistinguishable from AP in enzymatic properties and stability.
Keywords: alkaline phosphatase/extracellular secretion/repetitive sequences/type I secretion system/ß-roll structure
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionAcknowledgementsREFERENCES |
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Biotechnological applications mostly rely on the production of recombinant proteins, which can be achieved by a prokaryotic, eukaryotic or cell-free system. Although eukaryotic and cell-free systems have recently gained enormous attention because of their usefulness for production of eukaryotic proteins in functional form, prokaryotic system, especially an Escherichia coli system, is still generally preferable because of its simplicity and extensive genetic support. Unfortunately, intracellular production of foreign proteins in E.coli often results in accumulation of non-functional proteins, due to aggregation or misfolding, or rapid degradation of these proteins by intracellular proteases. Several techniques have been developed to overcome these problems, such as co-expression with molecular chaperones (Baneyx, 1999
) and extracellular production of proteins (Choi and Lee, 2004). Proteins secreted to the external medium are protected from aggregation and can fold into a native structure, because their concentrations are extremely reduced once they are secreted to the external medium. They are also protected from proteolytic degradation, because Gram-negative bacteria secrete few proteases to extracellular medium (Swamy and Goldberg, 1982). Furthermore, they can be easily purified from the culture supernatant without disrupting the cells.
There are at least five extracellular secretion systems in Gram-negative bacteria that have been characterized, many of which have been successfully reconstructed in E.coli (Omori and Idei, 2003; Choi and Lee, 2004). Type I secretion system (TISS) is one of these systems. Secretion by this system occurs in a single, energy-coupled step without any periplasmic intermediate (Andersen, 2003). This secretion machinery consists of three protein subunits—ATP-binding cassette (ABC) protein, membrane fusion protein (MFP) and outer membrane protein (OMP). These subunits form a conduit for protein secretion, protruding the inner and outer membranes of Gram-negative bacteria (Andersen, 2003; Omori and Idei, 2003). Most passenger proteins secreted by this system have a C-terminal secretion signal that remains intact after secretion and several repeats of a nine-residue sequence motif, GGxGxDxux (x: any amino acid, u: hydrophobic amino acids), located upstream of the secretion signal, thus are called RTX (repeats in toxin) family proteins (Andersen, 2003). These repetitive sequences are known to form a ß-roll structure upon Ca2+ binding (Baumann et al., 1993).
Family I.3 lipases are characterized by their ability to be secreted by TISS (Lip system) (Arpigny and Jaeger, 1999). We have previously shown that family I.3 lipase from Pseudomonas sp. MIS38 (PML) is effectively secreted from the E.coli cells carrying the Serratia marcescens Lip system to the extracellular medium (Kwon et al., 2002). PML is composed of 617 amino acid residues and, like most of RTX proteins, consists of the N-terminal catalytic domain and C-terminal domain containing a secretion signal and 12 repeats of a GGxGxDxux motif (Amada et al., 2000; Kwon et al., 2000). These repeats have been suggested to form a ß-roll structure upon Ca2+ binding (Amada et al., 2001) and to be required for intracellular stability, efficient secretion and functional structure of PML (Kwon et al., 2002; Angkawidjaja et al., 2005). These results encouraged us to examine whether the C-terminal domain of PML can be used as a secretion tag for extracellular production of heterologous proteins. Various proteins, including E.coli alkaline phosphatase (AP), have been reported to be secreted to the external medium from E.coli carrying authentic or heterologous TISS as a fusion protein with the C-terminal domain of E.coli haemolysin (Hly) (Mackman et al., 1987; Hess et al., 1990; Kenny et al., 1991; Hanke et al., 1992) or Erwinia chrysanthemi protease (Palacios et al., 2001). However, no biochemical studies have been conducted on these fusion proteins.
AP is a periplasmic protein that is secreted in a Sec-dependent manner. Transport competency of AP depends on a 22-residue N-terminal signal peptide that is cleaved by a membrane-bound periplasmic signal peptidase after translocation through the inner membrane (Inoyue et al., 1982). In the present study, we showed that AP is efficiently secreted to the external medium from E.coli cells carrying the S.marcescens Lip system as a fusion protein with a C-terminal domain of PML. Purification and biochemical characterization of this fusion protein indicated that this fusion protein is functionally indistinguishable from AP without a tag.
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Materials and methods |
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Cells and plasmids
The Escherichia coli strains used were DH5 [F–, hsdR17(rK–, mK+), recA1, endA1, deoR, thi–1, supE44, gyrA96, relA1] and JM109 [e14–(McrA–) recA1 endA1 gyrA96 thi–1 hsdR17 (rK–, mK+) supE44 relA1 
(lac-proAB) (F' traD36 proAB lacIqZM15)]. Plasmids pYBCD20 harboring the lipBCD gene from S.marcescens SM8000 (Kawai et al., 1998), pET-PML for overproduction of PML (Amada et al., 2000) and pET-PML5 for overproduction of PML5 (Kwon et al., 2002) were constructed previously. Plasmids pUC18 and pBlueScript II SK(+) were obtained from Toyobo.
Materials
AP was obtained from Wako Pure Chemical. p-Nitrophenyl phosphate (pNPP), oxidized form of glutathione (GSSG) and reduced form of glutathione (GSH) were obtained from Sigma. T4 polynucleotide kinase (PNK) and T4 DNA ligase were obtained from Takara.
Plasmid construction
E.coli JM109 strain was used for construction of all plasmids. For construction of plasmid pUC-PMLC, the gene encoding PMLC was amplified by PCR using 5'-TTGTTTCTAGATAAGAAGGAGTTGGATCC ATGGGCAGTGACAGTAATG-3' (primer 1) and 5'-CTTCTAAGCTTTCAGGCGATCACAATTC-3' (primer 2), where underlined bases represent XbaI and BamHI sites for primer 1 and HindIII site for primer 2. Plasmid pET-PML was used as a template. The resulting DNA fragment was digested with XbaI and HindIII and ligated to the corresponding sites of pUC18 to generate pUC-PMLC. In this plasmid, transcription of the gene encoding PMLC is under the control of the lac promoter, and two BamHI sites are closely located with each other, such that the XbaI site is located between these sites.
For construction of plasmid pUC-AP-PMLC, the gene encoding AP without its own N-terminal signal peptide was amplified by PCR using primer 3 (5'-GTTTCTAGAAAGAAGGAGATATACATATGACACCAGAAATGCCTG-3') and primer 4 (5'-TTTTTGGATCCTTTCAGCCCCAGAGCGGCT-3'), where underlined bases represent XbaI site for primer 3 and BamHI site for primer 4. The genomic DNA of E.coli, which was prepared from a Sarkosyl lysate as previously described (Imanaka et al., 1981), was used as a template. The resulting DNA fragment was digested with XbaI and ligated to the XbaI-SmaI sites of pBlueScript II SK(+) to generate pBS-AP. In this plasmid, the HindIII site is located downstream of the BamHI site, which is substituted for the stop codon of the AP gene. Plasmid pUC-PMLC was digested with BamHI and HindIII and the small BamHI-HindIII fragment was ligated to the corresponding sites of pBS-AP. The resultant plasmid pBS-AP-PMLC was then digested with XbaI and HindIII, and the small XbaI-HindIII fragment was ligated to the corresponding sites of pUC18.
For construction of plasmids pUC-AP-PML5C and pUC-AP-PML0C, the genes encoding PML5C and PML0C were amplified by PCR using plasmids pET-PML5 and pET-PML as the template, respectively. The primers used to amplify these genes were primers 1 and 2 for PML5C and primer 5 (5'-GGGAATTCATGGGATCCGGTTTTACTCG-3') and primer 2 for PML0C, where underlined bases represent the BamHI site. The amplified DNA fragments were digested with BamHI and HindIII and ligated to the corresponding sites of pBS-AP. The resultant plasmids pBS-AP-PML5C and pBS-AP-PML0C were then digested with XbaI and HindIII and the small XbaI-HindIII fragments were ligated to the corresponding sites of pUC18.
For construction of plasmid pUC-AP, the gene encoding AP was amplified by PCR using primer 3 and primer 6 (5'-TTTTTAAGCTTTCATTTCAGCCCCAGAGC-3'), where underlined bases represent the HindIII site. Plasmid pBS-AP was used as a template. The resultant DNA fragment was digested with XbaI and HindIII and the small XbaI-HindIII fragment was ligated to the corresponding sites of pUC18.
PCR was performed in 25 cycles using a thermal cycler (Gene Amp PCR System 2400; Perkin-Elmer) and KOD DNA polymerase (Toyobo). The nucleotide sequence was confirmed with an ABI PRISM 310 Genetic Analyzer (Perkin-Elmer). All DNA oligomers for PCR were synthesized by Hokkaido System Science.
Protein secretion
Escherichia coli DH5 cells were transformed with pYBCD20 and pUC18 derivatives. The transformants were grown in LB medium containing 50 mg/l ampicillin and 30 mg/l chloramphenicol at 30°C for 24 h with constant shaking. The culture was then centrifuged at 15 000 r.p.m. for 10 min or 8000 r.p.m. for 30 min at 4°C to separate the cells and supernatant. The proteins accumulated inside the cells and secreted to the external medium were analyzed by 15% SDS–PAGE (Laemmli, 1970). To estimate the amount of these proteins, the gel image was analyzed by Scion Image program (http://www.scioncorp.com). The N-terminal amino acid sequence of the secreted protein was determined by a pulse-liquid automated sequencing system Procise 491 (Perkin-Elmer).
Protein purification
PMLC and AP-PMLC were collected from the culture supernatant by 80% ammonium sulfate precipitation. The protein pellets were dissolved in 20 mM Tris–HCl (pH 8.0) containing 10 mM EDTA, 0.5 M arginine, 5% glycerol and 10 mM dithiothreitol, followed by dialysis against 20 mM Tris–HCl (pH 8.0) containing 5% glycerol, 1 mM GSSG and 0.2 mM GSH. The resultant solution was then applied to a column of DE52 (Whatman), which was equilibrated with 20 mM Tris–HCl (pH 8.0). The protein was eluted from the column by linearly increasing the NaCl concentration from 0 to 0.5 M. The fractions containing the protein were collected, concentrated using Centricon YM10 (PMLC) or YM50 (AP-PMLC) (Millipore), and applied to a column of HiLoad16/60 Superdex 200 (Amersham Biosciences), which was equilibrated with 20 mM Tris–HCl (pH 8.0) containing 0.15 M NaCl and 5% glycerol (PMLC) or the same buffer containing 10 mM MgCl2 and 1 mM ZnSO4 (AP-PMLC), for gel filtration. The fractions containing the protein were collected and used for biochemical characterizations. The protein concentration was determined from UV absorption using an A280 value of 0.66 for 0.1% solution for both proteins, which was calculated based on the amino acid sequence of the protein (Gill and von Hippel, 1989).
Enzymatic activity
The AP activity was determined using pNPP as a substrate. The reaction mixture (1 ml) contained 0.5 M Tris–HCl (pH 8.5), 10 mM MgCl2, 1 mM ZnSO4 and 5 mM pNPP. The enzymatic reaction was carried out at 60°C for 10 min and terminated by the addition of 50 µl of 4 M NaOH. The amount of p-nitrophenol (pNP) produced by the reaction was determined from the molar absorption coefficient value of 16 200 M–1cm–1 at 410 nm. One unit (U) of enzymatic activity was defined as the amount of the enzyme that produces 1 µmol of pNP per min. The specific activity was defined as the enzymatic activity per milligram or µmol of the protein.
DNA dephosphorylation
Dephosphorylation of plasmid DNA by AP-PMLC was analyzed as described by Rina et al. (2000). The plasmid vector pBlueScript II SK(+) was digested with EcoRI. Following phenol/chloroform extraction and ethanol precipitation, 1.5 U of AP-PMLC was added to the digested DNA and the mixture was incubated in AP reaction buffer [0.5 M Tris–HCl (pH 8.5) containing 10 mM MgCl2 and 1 mM ZnSO4] for 30 min at 60°C, in a total reaction volume of 1 ml. After inactivation of AP-PMLC with phenol/chloroform extraction, the dephosphorylated DNA was collected by ethanol precipitation and the ligation efficiency of its ends was tested. For re-phosphorylation of DNA, the dephosphorylated DNA was precipitated with ethanol and re-dissolved in PNK assay buffer [50 mM Tris–HCl (pH 8.0) containing 10 mM MgCl2 and 5 mM dithiothreitol]. PNK and ATP were added to the sample and the reaction was carried out at 37°C for 30 min. The ligation was carried out at 16°C for 2 h using T4 DNA ligase and ATP.
CD spectra
The far-UV CD spectra of the protein were measured at 25°C in 20 mM Tris–HCl (pH 8.0) containing 5% glycerol, 10 mM MgCl2 and 1 mM ZnSO4 in the presence or absence of 10 mM CaCl2 on a J-725 automatic spectropolarimeter (Japan Spectroscopic Co.). The protein concentration was 0.07–0.1 mg/ml and the optical path length was 2 mm. The mean residue ellipticity [
], which has the units of deg cm2 dmol–1, was calculated by using an average amino acid molecular weight of 110.
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Results and discussion |
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Design of fusion protein
PML contains 12 repeats of a nine-residue sequence motif at residues 373–557. We have previously shown that the C-terminal 19 residues are responsible for secretion of PML, while repetitive sequences are not (Kwon et al., 2002). However, the repetitive sequences are important for efficient secretion of PML, because secretion levels of the deletion mutants of PML, PML5 and PML0, which contain five and no repetitive sequences, are reduced by 2- and 40-fold, respectively, as compared with that of PML. Therefore, to examine whether the repetitive sequences are important for secretion of heterologous proteins as well, three fusion proteins were designed, such that the C-terminal domain of PML containing 0 (PML0C), 5 (PML5C) or all 12 (PMLC) repetitive sequences is attached to the C-terminus of AP. The amino acid sequences of the resultant fusion proteins AP-PMLC, AP-PML5C and AP-PML0C, as well as those of PMLC and AP, are schematically shown in Fig. 1.
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Secretion of proteins
Protein secretion was examined using E.coli DH5 cells carrying the S.marcescens Lip system (Kawai et al., 1998). Both genes encoding fusion protein and Lip system are constitutively expressed in these cells. Separation of the culture supernatant and whole cell extract, followed by SDS–PAGE analysis, indicated that all three fusion proteins (AP-PMLC, AP-PML5C and AP-PML0C) were efficiently secreted to the external medium, although the secretion level of the protein slightly decreased as the number of the repetitive sequences decreased (Fig. 2). The cellular contents of these fusion proteins were too low to be detected by staining with Coomassie Brilliant Blue (CBB), suggesting that none of these fusion proteins accumulates inside the cells or that the fusion protein accumulated inside the cells are rapidly degraded by intracellular proteases. The secretion levels of the proteins were estimated to be 10, 7.7 and 7.5 mg/l culture for AP-PMLC, AP-PML5C and AP-PML0C, respectively, from the intensity of the band visualized with CBB staining. The secretion of these proteins was dependent on the Lip system, because AP-PMLC was not secreted to the external medium from E.coli DH5 cells, which do not carry the Lip system (Fig. 2, lane 2). Likewise, the secretion of AP to the extracellular medium was promoted by a secretion tag (PMLC, PML5C or PML0C), because AP without this tag was not secreted to the external medium from E.coli DH5 cells carrying the Lip system (Fig. 2, lane 6). When the secretion tag alone was examined for secretion using E.coli DH5 cells carrying the Lip system, only PMLC was efficiently secreted to the external medium (data not shown). Its secretion level was 18 mg/l culture. PML5C and PML0C were neither secreted to the extracellular medium, nor accumulated inside the cells, suggesting that these proteins are rapidly degraded by intracellular proteases upon synthesis.
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Purification of PMLC and AP-PMLC
PMLC and AP-PMLC were purified to give a single band on SDS–PAGE from the culture supernatant of E.coli DH5 cells harboring plasmids pYBCD20 and pUC18 derivatives (data not shown). The amount of the protein purified from 1 l culture was 3.6 mg for PMLC and 2.7 mg for AP-PMLC. Determination of the N-terminal amino acid sequences of these proteins indicated that the initiator methionine residues, attached to the N-termini of PMLC and AP-PMLC, were partially (45%) removed and were not removed, respectively, during overproduction and purification. The molecular masses of PMLC and AP-PMLC were estimated to be 29 and 72 kDa, respectively, from SDS–PAGE. These values are comparable to those calculated from their deduced amino acid sequences (26.7 kDa for PMLC and 72.3 kDa for AP-PMLC). The molecular masses of PMLC and AP-PMLC were also estimated to be 27 and 146 kDa, respectively, from gel filtration chromatography, suggesting that PMLC exists as a monomer, while AP-PMLC exists as a dimer.
It is noted that the amount of AP-PMLC purified from 1 l culture was reduced by 25 times when the protein was not treated with a glutathione redox buffer. This result suggests that AP-PMLC secreted to the extracellular medium cannot efficiently fold into its dimeric and active structure, due to incorrect formation of disulfide bonds. In fact, it has been reported that AP requires Dsb (disulfide-bond isomerase) proteins for in vivo folding (Rietsch et al., 1996; Bolhuis et al., 1999) and periplasmic fraction or GSSG for in vitro folding (Akiyama and Ito, 1993).
Enzymatic activity of AP-PMLC
The pH (Fig. 3, panel A) and temperature (Fig. 3, panel B) dependencies of the AP activity of AP-PMLC were nearly identical with those of AP. The stability of AP-PMLC against heat inactivation was also nearly identical with that of AP (Fig. 3, panel C). Furthermore, the specific activity of AP-PMLC, which was determined to be 5300 ± 230 U/µmol (37 ± 1.6 U/mg), is highly similar to that of AP, which was determined to be 5500 ± 200 U/µmol (58 ± 2.0 U/mg). These results indicate that PMLC attached to the C-terminus of AP as a secretion tag does not seriously affect the structures and functions of AP.
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Dephosphorylation of DNA by AP-PMLC
To examine whether AP-PMLC removes 5'-terminal phosphates from DNA fragments, plasmid Bluescript II SK(+) was digested with EcoRI, treated with AP-PMLC and ligated with T4 DNA ligase. As shown in Fig. 4, the linearized plasmid DNA was circularized, oligomerized or both upon ligation, but was not when it was pre-treated with AP-PMLC (Lanes 2 and 3). This linearized and dephosphorylated plasmid DNA could be circularized, oligomerized or both upon ligation when it was pre-treated with PNK for re-phosphorylation (lanes 4 and 5). These results indicate that AP-PMLC has an ability to remove 5'-terminal phosphates from DNA fragments, as does AP.
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phage; lane 1, plasmid DNA linearized with EcoRI; lane 2, ligation product of linearized DNA; lane 3, ligation product of linearized DNA dephosphorylated with AP-PMLC; lane 4, ligation product of linearized DNA re-phosphorylated with PNK; lane 5, EcoRI digest of the final ligation product. Numbers along the gel represent the sizes (kbp; kilobase-pairs) of individual HindIII fragments of CD spectra
It has previously been shown that the far-UV CD spectrum of PML is significantly changed upon Ca2+ binding, probably due to formation of ß-roll structure (Amada et al., 2001). To examine whether the repetitive sequences attached to AP form a ß-roll structure as well, the far-UV CD spectrum of AP-PMLC was measured in the presence and absence of Ca2+. The spectrum of PMLC was also measured as a reference. All spectra were measured in the presence of Mg2+ and Zn2+, because these metal ions are required not only for enzymatic activity of AP, but also for stabilization of dimeric structure of AP (Reynolds and Schlesinger, 1969; Anderson et al., 1975). The far-UV CD spectrum of PMLC was not seriously changed in the presence of these metal ions (data not shown). However, this spectrum was considerably changed in the presence of Ca2+ (Fig. 5, panel C), suggesting that the repetitive sequences retain an ability to form a ß-roll structure even when the N-terminal domain is removed. The spectrum of PMLC in the presence of Ca2+ was similar to a typical one for ß-structure (Greenfield and Fasman, 1969). A similar spectral change has been observed for CyaA (adenylate cyclase) (Rose et al., 1995).
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The far-UV CD spectrum of AP was not seriously changed in the presence of Ca2+ (Fig. 5, panel A). The spectrum of AP-PMLC was also not significantly changed in the presence of Ca2+, although it was slightly changed such that the depth at 208 nm decreases and the depth around 220 nm increases (Fig. 5, panel B). When AP and PMLC were mixed with a molar ratio of 1 : 1, a significant spectral change was observed in the presence of Ca2+ (Fig. 5, panel D). Like the spectrum of AP-PMLC, the depth at 208 nm decreases and the depth around 220 nm increases. These results suggest that the repetitive sequences of AP-PMLC are partially folded into a ß-roll structure. Folding of AP into a functional dimeric structure may interfere with the formation of a ß-roll structure in this fusion protein.
Role of GGxGxDxux repetitive sequences in the fusion protein
It has been reported for various passenger proteins of TISS that the GGxGxDxux repetitive sequences are important for their intracellular stability and/or functionality (Felmlee and Welch, 1988; Letoffe and Wandersman, 1992; Miyajima et al., 1998; Bejerano et al., 1999; Kwon et al., 2002; Angkawidjaja et al., 2005). These repeats play indirect role in the secretion efficiency of the natural passenger proteins by conferring their intracellular stability. We have previously shown that the secretion levels and stabilities of the PML derivatives with less than four repeats are greatly reduced as compared with those of PML (Kwon et al., 2002). Upon complete removal of the repeats, the secretion level of PML is reduced by 40-fold. These results suggest that the repeats are folded into a ß-roll structure intracellularly and thereby contribute to increase intracellular stability and secretion efficiency of passenger proteins. However, the repeats were not so important for secretion of AP-PMLC, because the secretion level of the fusion protein was reduced only by 25% upon complete removal of the repeats (Fig. 2). Similar results have been reported for HlyA. The repetitive sequences of HlyA are important for functional conformation and efficient secretion of HlyA (Felmlee and Welch, 1988), but not for secretion of the AP-HlyA fusion protein (Hess et al., 1990).
Many proteins that are subjected to membrane translocation are thought to be intracellularly unfolded or partially folded. These proteins require molecular chaperones to protect them from proteolytic degradation and/or aggregation (De Keyzer et al., 2003). In fact, HasA (heme-acquisition A), which does not contain any repetitive sequence but is secreted by TISS, requires SecB for its efficient secretion (Sapriel et al., 2002). SecB is known as a molecular chaperone (Kumamoto, 1991). However, none of the passenger proteins of TISS, which contain the repetitive sequences, has been reported to require molecular chaperones (Blight and Holland, 1994). These results suggest that the repetitive sequences function as a molecular chaperone. Although, it has been reported that AP does not require SecB for normal transport via Sec system (Kumamoto and Beckwith, 1983), data so far accumulated suggest that AP interacts with SecB intracellularly (Derman et al., 1993; Kononova et al., 2001). Therefore, AP-PMLC and AP-HlyA do not require repetitive sequences for efficient secretion via TISS, probably because SecB functions as a substitute for the repetitive sequences.
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Conclusion |
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In the present study, we showed that AP with a C-terminal secretion tag is efficiently secreted to the external medium from the E.coli cells carrying a heterologous TISS. Purification and biochemical characterization of this protein indicate that the C-terminal secretion tag does not seriously affect the structures and functions of AP. Extracellular production of recombinant proteins can be achieved using Gram-positive bacteria, such as Bacillus subtilis (Ohmura et al., 1984
) and E.coli strain with leaky outer membrane (Atlan and Portalier, 1984, 1987). However, the extracellular production system using TISS and Gram-negative bacteria is advantageous over these systems, because of its high selectivity (Gram-negative bacteria secrete few proteins to extracellular medium). It is noted that the fusion proteins were not designed, such that a C-terminal secretion tag can be removed from the fusion proteins, in this study. However, it would be possible to remove it from the fusion proteins, if the cleavage sites for sequence-specific proteases, such as factor Xa and thrombin, are inserted between the proteins of interest and a C-terminal secretion tag.
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Footnotes |
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Edited by Taiji Imoto
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Acknowledgements |
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The authors thank Drs H.-J. Kwon, M. Haruki and M. Morikawa for the helpful discussions. This work was supported in part by a Grant-in-Aid for National Project on Protein Structure and Functional Analyses and by a Grant-in-Aid for Scientific Research (No. 16041229) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Received February 19, 2006; revised March 20, 2006; accepted March 29, 2006.
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