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Journal of General Microbiology (1993), 139, 2465-2473. Printed in Great Britain 2465 Targeting of interleukin-2 to the periplasm of Escherichia coli GABRIELE HALFMANN,'t HERVEBRAILLY,~ ALAINBERNADAC, FELIX A. MONTEROJULIAN,~ CLAUDE LAZDUNSKI' and DANIELBATY~* 'Laboratoire dlngknierie et Dynamique des S y s t h e s Mernbranaires du CNRS, 31 chemin J. Aiguier, BP 71, 13402 Marseille Cedex 20, France 'lmmunotech SA, 130 Avenue De Lattre De Tassigny, B.P. 177F-13276 Marseille Cedex 9, France (Received 10 February 1993; revised 27 April 1993; accepted 10 May 1993) A synthetic gene coding for interleukin-2 (IL-2) was used to produce large amounts of recombinant IL-2 (met-IL2) in Escherichia cofi. Met-IL-2 was found to accumulate in the cytoplasm in an insoluble, aggregated form. Inclusion bodies located at the pole caps of cells were detected using immunogoldlabelling. Constructs were designed to fuse the IL-2 gene to DNA fragments encoding signal peptides for an outer-membrane protein (OmpA) or for a periplasmic protein (PhoA) of E. coli. No significant maturation was observed with these fusion proteins which were found in an insoluble form in the cytoplasm. The influence of charge disposition at the N-terminus of the mature portion of the protein was investigated by replacing positively charged amino acids with glutamic acid. None of the introduced substitutions had any effect. Various factors that might affect expression, secretion and folding were examined in an attempt to obtain secretion. By fusing IL-2 to the precursor maltose-binding protein (preMBP) a large fraction of the preMBP-IL-2 protein was correctly processed and transported to the periplasmic space. IL-2 derived from MBP-IL-2 after FXa cleavage possessed similar specific activity to recombinant IL-2 produced in Chinese Hamster ovary cells. Introduction The lymphokine IL-2 plays a central role in the development of the immune response. It is an inducible protein synthesized and secreted by activated T lymphocytes with the N-terminal extension of a signal peptide. As a growth factor, IL-2 acts via a specific receptor and stimulates the proliferation of IL-2-dependent T cells (Taniguchi et al., 1983; Smith, 1988). Interest in IL-2 has increased with the finding that this lymphokine can be used in the treatment of solid tumours (Rosenberg et al., 1986) and for partial restoration of T-cell function (Pahwa et al., 1989). The cloning and synthesis of human IL-2 in E. coli was first described by Devos et al. (1983), but most of the * Author for correspondence. Tel. + 33 91 164117; fax + 33 9 1712124; e-mail [email protected]. t Present address : Universitat Wien, Institut fur Mikrobiologie und Genetik, Althanstr. 24, A- 1090 Wien, Austria. Abbreviations : IL-2, interleukin-2 ; met-IL-2, recombinant interleukin-2 ; MBP, maltose-binding protein. protein produced remained insoluble in the cytoplasm. Biological activity therefore could not be restored completely and was not proportional to total synthesis. Expression of recombinant proteins in E. coli has become a valuable tool for basic research and industrial applications. However, high levels of expression of eukaryotic proteins lead in most cases to the formation of insoluble inclusion bodies in E. coli (Marston, 1986). The extraction, denaturation and renaturation of these proteins in their active form are time-consuming and expensive. The efficiency of renaturation and the activity of the renaturated protein depend on the correct formation of its disulphide bridges (Marston, 1986). Expression via secretion offers several advantages over intracellular expression. If the signal sequence is processed correctly, the N-terminus of the recombinant protein will be identical to the authentic product. Export of proteins to the periplasm can prevent the degradation of the polypeptide. Disulphide bond formation in E. coli has been shown to occur concomitantly with export from the cytoplasm (Pollit & Zalkin, 1983; Derman & Beckwith, 1991). Folded eukaryotic proteins with correct disulphide bonds can therefore be produced (Oka et al., 1985; Parker & Wiley, 1989),which is a major advantage. 0001-8143 0 1993 SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 03 Aug 2017 19:13:35 2466 G . Halfmann and others In some cases the export of heterologous proteins to the periplasm of E. coli requires only the correct fusion between a prokaryotic signal peptide and the mature portion of the foreign protein provided that the protein itself is normally secreted (Ghrayeb et al., 1984; Duffaud et al., 1987; Better et al., 1988; Parker & Wiley, 1989). Some eukaryotic proteins, such as interleukin-lp, can be recovered by simple osmotic shock (Joseph-Liauzun et al., 1990); others, however, are unable to enter the export pathway. For these proteins, additional steps must be taken to obtain their secretion. IL-2 was used as a model protein to investigate the possible export of a protein known to form insoluble particles in the cytoplasm. This study of the export of IL2 in E. coli represents a continuation of a systematic analysis of different proteins to determine the prerequisites for the export of heterologous proteins. The influence of prokaryotic signal sequences has been investigated for several proteins (Denkfle et al., 1989; D. Baty, unpublished results). In this study, different factors influencing protein maturation and export, such as (i) the nature of the signal peptide per se (OmpA or PhoA), (ii) the role of charge distribution near the leader peptidase cleavage site, (iii) the influence of chaperones (GroES and GroEL), (iv) the incubation temperature and inducer concentration, (v) the use of lysis proteins and (vi) fusion to a known, secreted protein (preMBP) efficiently targeted by SecB to the export apparatus, were investigated. Methods promotor regulated by the lacIQrepressor. The 420 bp EcoRI-Hind111 fragment of BBG3 replicative form (BBL) was inserted between the EcoRI and HindIII sites of pJF118EH to create pGF25 (Fig. 1). The ssOmpA-IL-2 fusion was created by linking the synthetic IL-2 gene to the signal peptide of OmpA by inserting the XhoI-BglII fragment of BBG3 between the BamHI and HindIII sites of PIN-111-ompA-Hind (Rentier-Delrue et al., 1988) connected by the oligonucleotides 5'AGCTGCTCCTACTAGC-3' and 5'-TCGAGCTAGTAGGAGC-3' to create pFQ (Fig. 2). The ssPhoA-IL-2 fusion in pFP6 was constructed by inserting the XhoI-Hind111 fragment of BBG3 between the HindIII sites of plasmid pBX4.1 (D. Baty, unpublished result) connected by the oligonucleotide cassette used for the previous construction (Fig. 2). The XhoI-Hind111 fragment of BBG3 was inserted between the StuI and HindIII sites of pMal-p (New England Biolabs) using the oligonucleotides 5'-GCTCCTACTAGC-3' and 5'TCGAGCTAGTAGGAGC-3' to give pLS28. Gene expression andprotein characterization.The plasmid-containing strains were grown in rich medium at the indicated temperatures. Gene expression was induced by the addition of 01-1 m~ -IPT G . Cell fractionation (Neu & Heppel, 1965) was performed as follows : cells in late exponential growth were harvested and washed in TE (10 mMTris/HCl pH 7.5, 1 mM-EDTA). After centrifugation, the cell pellet was gently resuspended in 30 mM-Tris/HCl, pH 7.5, 20 YO (w/v) sucrose, 0.5 mM-PMSF, 1 mM-EDTA to a density of 10" bacteria ml-'. After 10 min at room temperature, the suspension was centrifuged for 5 min at 6000g. The sucrose solution was carefully drained from the tube and the pellet was resuspended in a same volume of cold water. The resuspended cells remained on ice for 10 min and were centrifuged at 15000g for 10 min at 4 "C. The supernatant is referred to as osmotic shock fluid (periplasmic fraction). The pellet was resuspended in 30 mM-Tris pH 7.5, 0.5 mM-PMSF, 1-25mM-MgCl,, 25 pg DNAase I ml-l, freeze-thawed six times and centrifuged for 5 min at 15000g. The pellet and the supernatant are referred to as the membrane and cytoplasmic fraction, respectively. All samples were resuspended in SDS loading buffer [60 mM-Tris/HCl pH 6.8, 1 % (w/v) SDS, 10% Hinm HindIII Strains and media. E. coli TB1 araA(1ac proAB) rpsL (#80 lac2 AM15) (Johnston et al., 1986) was used as host for the expression of the MBP-IL-2 fusion protein. In all other experiments, the plasmids were expressed in E. coli W3110 (Lloub6s et al., 1986). M9 minimal medium and LB media have been described by Miller (1972). Antibiotic resistant plasmids were maintained in transformed strains by addition of 100 pg ampicillin ml-' and, when required, chloramphenicol (50 pg ml-') or kanamycin (50 pg I&') to the growth medium. DNA manipulation. The synthetic gene encoding IL-2 was provided by BBL. Oligomers were synthesized using a model 380A automatic DNA synthesizer (Applied Biosystem). The nucleotide sequence analysis by the T7 polymerase chain termination method was carried out using bacteriophage M 13 or NaOH-denaturated plasmid templates [lo80 Ci (Messing, 1983; Hattori & Sakaki, 1986) and [cz-~~PI~ATP (40 MBq) mmol-' ; Amersham]. Site-directed mutagenesis was performed as described by Baty et al. (1987b). To exchange lysines (K) either at position K8 and/or K9 of IL-2 for glutamic acid (E), the following oligonucleotides were used : K8E, 5'-GAGTTTTCTCAGTGCTCG-3'; K9E, 5'-GTTGAGTTTCCTTAGTGCT-3'; K8/9E, 5'GTTGAGTITCCTCAGTGCT-3'. The substitution of cysteine 125 (C125) by alanine 125 (A125) was performed with the oligonucleotide C125A, 5'-CGATTGGGAAAGGTG-3'. Plasmid construction. The plasmid pJFl18EH (Furste et al., 1986) was used as a source to express IL-2 under the control of the tuc EcoRI v EcoIWHindIII HindUl ' 5680 bp Fig. 1. Construction of pGF25. Plasmid GF25 was constructed by inserting the EcoRI-Hind111 fragment (black) containing the coding sequence for IL-2 into pJFl18EH. The arrow indicates the direction of transcription from the tac promotor. The bla (white) and the lucP (stippled) genes are indicated. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 03 Aug 2017 19:13:35 Targeting of IL-2 to periplasm of E. coli 2467 HindIII I \r/ HindIIVEamHI Oligonucleotides forjunction pBX4- I \/ Hind111 XhoI tac [ XhoI PFP6 5370bp MKQSXIALUWLLTTPVTKAAPTSSSTKKT... Fig. 2. Construction of the ssOmpA-IL-2 and ssPhoA-IL-2 fusion proteins. The sequences coding for the signal sequence of OmpA (bold dashed) and PhoA (bold dotted) are indicated. pBX4.1 contains the growth release factor (GRF) gene (dashed) which is removed during the construction. The other genes are as described in Fig. 1 except that lacl is used in place of lac14. The amino acid sequences of the signal sequences preceding the IL-2 sequence are indicated in bold at the bottom of the figure. (v/v) glycerol, 2 % (v/v) /3-mercaptoethanol] heated to 95 "C for 5 min, analysed by 14 YOSDS-PAGE and immunoblotted on nitrocellulose as described by Towbin et al. (1979). The met-IL-2 and the IL-2 fusion proteins were visualized by immunodetection with monoclonal antibodies directed against IL-2. Pulse-chase experiments. E. coli W3110 harbouring pFQ (preOmpAIL-2) or pFP6 (prePhoA-IL-2) were co-transformed with plasmid groES/EL (Goloubinoff et al., 1989). Pulse-chase experiments were performed by diluting overnight cultures of the samples 100-fold in M9 medium. The presence of both plasmids was maintained by the addition of ampicillin (100 pg ml-') and chloramphenicol(50 pg ml-I). At an OD, of 0 4 simultaneous induction of the IL-2 fusions and GroES/GroEL was obtained by the addition of 1 mM-IPTG. After 10 min, 10 pCi (370 kBq) [35S]methionine was added. After a 20 s pulse label, the culture was chased for varying periods of time with 1 % unlabelled methionine. Aliquots corresponding to 60000 c.p.m. were loaded on a 14% SDS-polyacrylamidegel. IL-2 bioassay. The biological activity of human IL-2 was tested using the IL-2-dependentmurine T lymphocytecell line CTLL-2 (Gillis et al., 1978). The CTLL cell line was cultured in RPMI 1640 (Gibco) containing 1 mwpyruvate, 2 a-L-glutamine, 50 pg streptomycin and penicillin ml-' (all chemicals were from Flow), 10 % (v/v) foetal calf serum (Gibco) and supplemented with 1 ng rhIL-2 ml-' (Sanofi). The I L 2 bioassay was then performed as described previously. Briefly, 100 pl per well of the cell suspension at lo5 cells ml-' giving lo4 cells per well was added to 96-well culture plates together with 100 p1 per well of the IL-2 containing samples. The cells were cultured for 48 h at 37 "C with 5 % (v/v) CO, in a humidified incubator and were then pulsed for 8 h with 0-5 pCi (18.5 kBq) [3H]thymidine(Amersham) per well. The cells were then harvested and the radioactivity incorporated in the cell pellets was determined in a beta counter (Kontron). All determinations were performed in triplicate. A standard range of serial dilutions of calibrated IL-2 (Sanofi)was included in every experiment. One Unit of activity was defined as the amount of IL-2 that leads to half maximum proliferation of CTLL cells. Concentrations of IL-2 in the samples were then expressed in Units. In parallel with bioactivity determinations,IL2 concentrations were determined using a commercial immunoassay (Immunotech) calibrated against the international standard NIBSC 86/564. From these determinations specific activities, expressed as CTLL Units per ng IL-2, were derived for the different preparations. Electron microscopy. The detailed procedure for fixation, embedding, cryosectioning and staining has been described previously (Bernadac 8z Lazdunski, 1981). The only difference was the use of the monoclonal antibody directed against IL-2 and of gold-coated protein A instead of ferritin-labelled anti-rabbit antibodies. MBP-IG2 puriJication. The protocol used for the purification of MBP-IL-2 and its cleavage by FXa was that described by New England Biolabs with some modifications. The amylose column was washed Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 03 Aug 2017 19:13:35 2468 G. Halfmann and others with 50 mM-Tris/HCl, pH 7.5, 150 mM-NaC1 (TNA buffer). The periplasmic shock fluid was circulated through the column overnight at 60 ml h-' with TNA. The column was then washed with TNA until the A,,, reached the base line. The MBP-IL-2 was eluted with TNA containing 5 mmmaltose. maturation were examined by analytical cell fractionation. The OmpA-IL-2 and PhoA-IL-2 constructs showed a level of synthesis similar to that of the wild- Results High-level expression of mature IL-2 from pGF25 (Figs 1 and 3) resulted in its aggregation into inclusion bodies that were located either in the middle of the cells or at the polar caps (Fig. 5). This confirmed previous findings by Devos et al. (1983) that IL-2 aggregates in the cytoplasm of E. coli. To overcome this, we have investigated the targeting of IL-2 to the periplasm of E. coli by fusing it either to bacterial signal peptides or to the periplasmically-located maltose-binding protein. IL-2 was fused to two different E. coli signal sequences, one from the outer membrane protein (OmpA) and the other from the periplasmic alkaline phosphatase (PhoA) in pFQ and pFP6, respectively (Fig. 2). The synthesis of IL-2 and of the fusion proteins was compared in the same E. coli W3 110 strain under identical experimental conditions. About 5 YOof total cellular protein was expressed as metIL-2 or ssOmpA-IL-2 and 15% as ssPhoA-IL-2 (Fig. 3). The induced E. coli pGF25 (met-IL-2), pFQ (ssOmpAIL-2) and pFP6 (ssPhoA-IL-2) were fractionated and separated into spheroplasts, periplasmic and membrane fractions (Fig. 4). The hybrid proteins, ssOmpA-IL-2 (16.9 kDa) or ssPhoA-IL-2 (16.7 kDa), were neither properly processed to the mature form of IL-2 (14.5 kDa) nor secreted to the periplasm of E. coli (Fig. 4). The majority of the fusion proteins remained unprocessed in the membrane fraction. Only a weak processing of the precursor to the mature form could be observed (Fig. 4, arrows in lanes T). To localize the fusion proteins found in the membrane fraction, immunogold labelling was performed. Immunogold labelling of E. coli W3 110 revealed no difference in localization of the proteins of the cytoplasmic version or those derived from the ssPhoA-IL-2 (Fig. 5) or ssOmpA-IL-2 fusion proteins (not shown). These observations were consistent with the absence of processing of the fusion proteins and clearly demonstrated that they did not even enter the secretion pathway. The N-terminus of mature IL-2 has two consecutive positively charged lysines at positions 8 and 9 that may perturb the export process. To test this possibility, in vitro mutagenesis was performed to substitute the lysine residues (K) at positions 8 and 9 by glutamic acid residues (E) which should not influence the biological activity of IL-2 (Ju et al., 1987). K8, K9 or K8/9 were replaced by E8, E9 or E8/9, respectively. The mutant IL2 gene sequences were fused to the signal sequence of either OmpA or PhoA. Secretion competence and protein Fig. 3. Expression of met-IL-2, ssOmpA-IL-2 and ssPhoA-IL-2. IPTG (1 mM) was added to exponentially growing cultures of E. coli W3110 (pGF25, pFP6 or pFQ) at 37 "C when the OD, reached 1. At 0 , l and 3 h, samples were removed, centrifuged, resuspended in sample buffer, boiled for 5 min and aliquots corresponding to 0.2 OD,,, units, were run on 14% SDS-polyacrylamide gels. Proteins were blotted on nitrocellulose and immunoblot analysis was carried out using an IL-2 monoclonal antibody. The positions of prestained molecular mass standards (Gibco-BRL) are shown on the left. Fig. 4. Localization of the recombinant IL-2 and the IL-Zfusion polypeptides. The expression of cytoplasmic met-IL-2 (pGF25), ssOmpA-IL-2 (pFQ) and ssPhoA-IL-2 (pFP6) was induced in an exponentially growing culture at an OD,, of 1.0 by the addition of 1 mM-IPTG at 37 "C. Two hours after induction, samples were taken and cell fractionation was performed as described in Methods. Protein fractions corresponding to 0.2 OD,,, units were run on 14% SDSpolyacrylamide gels and immuno-detected with IL-2 antibodies. T (total cells), sl (supernatant of culture medium), s2 (sucrose fraction after osmotic shock), P (periplasmic fraction), C (cytoplasmic fraction) and M (membrane fraction). Arrows indicate the migration of mature IL-2. The positions of prestained molecular mass standards (GibcoBRL) are shown on the right. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 03 Aug 2017 19:13:35 Targeting of IL-2 to periplasm of E. coli 2469 Fig. 5. Localization of the cytoplasmic version of IL-2 (pGF25) and the prePhoA-IL-2 fusion by immuno-microscopy. E. coli W3 1lO(pGF25) and W3110(pFP6) were cultured in M9 medium at 37 "C to an ODao of 0.4.Cells were induced with IPTG for 4 h and then fixed with paraformaldehyde (2%, v/v) and glutaraldehyde (0.2%) for 1 h. Ultrathin sections were incubated with 20 pg IL-2 monoclonal antibodies ml-' for 60 min and labelled with rabbit anti-mouse serum and protein A-gold for 30 min. Uninduced and induced cells of pGF25 (a, b) and of pFP6 (c, d) are shown, respectively. Intracellular inclusion bodies could be seen in the cells even without any specific labelling of the proteins. Bar, 0.1 pm. type IL-2, as detected by immunoblotting (data not shown). None of the substitutions of lysine by glutamic acid (E8, E9 and E8/9) improved precursor maturation or translocation of the fusion proteins as compared to the wild-type (data not shown). IL-2 contains three cysteine residues, two forming a disulphide bridge. To assess whether the free cysteine residue 125 (C125), which is not essential for the biological activity of IL-2 (Ju et al., 1987) but may be responsible for the inter- and/or intramolecular formation of disulphide bridges, might be involved in the insolubility of the protein (and therefore increased protein aggregation), C125 was substituted by an alanine residue in the plasmids containing ssPhoA-IL-2 and ssOmpA-IL-2. C125 was also mutated, in addition to the replacement of the lysine residues by glutamic acid in the same plasmids. The expression was comparable to that of the wild type. No difference in protein maturation or translocation could be detected in the IL-2-Al25 mutants and their fusion derivatives (data not shown). C125 therefore seemed not to be the dominant factor in the formation of intracellular aggregates. We attempted to suppress premature folding and aggregation of the ssOmpA-IL-2 and ssPhoA-IL-2 fusion proteins that may have prevented translocation by increasing the intracellular level of the chaperones GroEL and GroES. Plasmids encoding groES and groEL (Goloubinoff et al., 1989) were introduced into E. coli W3 110 harbouring pFQ (ssOmpA-IL-2) or pFP6 (ssPhoA-IL-2). Simultaneous induction of the IL-2 fusions (ssOmpA-IL-2 or ssPhoA-IL-2) with groES and groEL did not result in any significant increase in the maturation of these protein fusions (data not shown). The pMal-p vector was used to construct a maltosebinding protein (MBP)-IL-2 fusion protein. This contains the sequence IEGR encoding the recognition site of the specific protease factor Xa, located inside the polylinker insertion site of the vector (Fig. 6a). After Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 03 Aug 2017 19:13:35 2470 G.HaZfmann and others Table 1. IL-2 activities in CTLL assay (4 SP FXa -)c lael wlE-IL-2 -+J, rmB bla ori Sample IL-2 bioactivity (CTLL; Units m1-l) IL-2 concn (ng m1-l) Specific activity (CTLL; Units ng-') 1 412 660 0 0.07 92 62 0 14-29 4.48 10.65 0 rhIL-2* MBP-IL-2t IL-2$ Bufferg * Recombinant Chinese Hamster ovary-derived IL-2. t MBP-IL-2 fusion protein purified by maltose affinity chromatography. $ IL-2 derived from MBP-IL-2 by FXa treatment. Q 10 mM-Tris/HCl, pH 7.5. proteolysis with factor Xa, the MBP-IL-2 fusion protein should be cleaved to give authentic IL-2 protein. Induction resulted in about 5 % of the total cellular protein being preMBP-IL-2 and MBP-IL-2 fusion proteins (Fig. 6 b, lanes T). Analytical cell fractionation revealed that most of the MBP-IL-2 hybrid protein was secreted to the periplasmic space of E. coli (Fig. 6 b, lanes P). The fusion protein was degraded during purification (Fig. 6 c , lane 0). After incubation with FXa, the hybrid proteins yielded MBP and IL-2 (Fig. 6c, lanes 4 and 8). The MBP-IL-2 fusion protein and the IL-2 derived from FXa cleavage were tested for their ability to sustain the proliferation of the IL-2-dependent cell line CTLL-2 (Table 1). Both recombinant proteins were found to be active in the assay. Interestingly, FXa cleavage yielded a protein with higher specific activity which was very similar to that of a preparation of Chinese Hamster ovary-derived recombinant IL-2 (rhIL-2). Discussion Fig. 6. Expression, secretion and purification of the MBP-IL-2 hybrid protein. (a) The IL-2 gene was inserted in the pMal vector that encodes the maltose-binding protein (MBP), the signal sequence (SS) of preMBP and the factor Xa cleavage site (IEGR). The vertical arrow heads indicate the sites of cleavage by the signal peptidase (SP) and the factor Xa (FXa), respectively. The horizontal arrows indicate the different relevant genes and promotors of the plasmid pLS28. (b) The preMBP-IL-2 hybrid was induced with 1 mM-IPTG in LB at 37 "C at an OD,, of 0-5.Two hours after induction, samples were taken and cell fractionation was performed as described in Methods. Protein samples corresponding to 0.5 OD,,, units were applied to a 14% SDSpolyacrylamide gel. After immunoblotting, the MBP-IL-2 fusion was visualized with anti-IL-2 and anti-MBP antibodies. Anti-/3-lactamase (anti-Bla) was used to control the quality of the periplasmic fraction. MBP-IL-2* represents a degradation product of MBP-IL-2. Lanes T (total cells), P (periplasmic fraction), C (cytoplasmic fraction) and M (n..mbrane fraction). The positions of prestained molecular mass standards (Gibco-BRL) are shown on the right. (c) After purification the preMBP-IL-2 protein was treated for 0, 4 or 8 h with FXa. After immunoblotting, the proteins were visualized with anti-IL-2. The positions of prestained molecular mass standards (Gibco-BRL) are shown on the left. In general, recombinant polypeptides can be accumulated to higher levels when retained intracellularly than when secreted. However, export of a polypeptide may be preferred for several reasons: (i) to allow formation of disulphide bridges required for function; (ii) to decrease proteolytic degradation, although severe proteolysis of heterologous proteins may occur in the periplasmic space ; (iii) to facilitate the purification of recombinant proteins. For these reasons, we tried to obtain an E. coli construct able to produce and export IL-2. The fusion of a prokaryotic signal peptide to the mature IL-2 did not promote export which contrasted with results observed for other recombinant proteins produced in E. coli (Ghrayeb et al., 1984; Better et al., 1988; Parker & Wiley, 1989; Denkfle et al., 1989). Not only, were the ssOmpA-IL-2 and the ssPhoA-IL-2 not exported but they did not even enter the export pathway and it is therefore likely that they aggregated very early after Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 03 Aug 2017 19:13:35 Targeting of IL-2 to periplasm of E. coli synthesis. The signal peptides may have become buried very soon after synthesis and were unable to direct the protein to the export machinery. By using IL-2 as a model polypeptide, we have investigated the effects of different factors that may interfere with the export of recombinant proteins. First we altered the charge distribution near the cleavage site since it had been demonstrated that by replacing basic residues in this region, defective export could be corrected (Summers & Knowles, 1989; Parker & Wiley, 1989). Although the mechanism of export in prokaryotes and eukaryotes appears to be similar, the presence of basic residues near the cleavage site is more frequent in eukaryotic secretory proteins (Watson, 1984; von Heijne, 1986). Whether this reflects some difference between early events in the secretory process of prokaryotes and eukaryotes remains an intriguing question. Expression and secretion of IL-2 in Streptomyces lividans resulted in active IL-2 being secreted to the culture supernatant with, however, inefficient processing and translocation of the precursor (Bender et al., 1990). The inability of the IL-2 fusions to enter the export pathway in E. coli may be explained mainly by thermodynamic considerations of the conformation of the mature region, independently of the signal sequence used. By contrast, experiments on the export of human interleukin-18 showed a remarkable influence of the bacterial peptide on the secretion of this eukaryotic protein (Denifle et al., 1989). Observations concerning the nature of the N-terminal residues of a protein as a key factor in secretion (for review, see Boyd & Beckwith, 1990) led us to introduce mutations in the non-secretable hybrid proteins that comprise the signal peptides of PhoA and OmpA fused to the complete IL-2 gene. Some of the mutations were designed to change the net charge near the N-terminus of the mature form of IL-2, while other alterations were made to avoid the incorrect formation of disulphide bridges (leaving the N-terminal sequences unchanged). We found that the substitution of the basic lysine residues by glutamic acid did not improve the secretion competence of the fusion proteins, regardless of the replacement of one or the other (K8 or K9) or both (K8/9) residues. Results obtained in studies of the influence of the net charge at the N-terminus of the mature form of prokaryotic proteins on protein export (MacIntyre et al., 1990; Yamane & Mizushima, 1988) therefore do not apply to the secretion of heterologous proteins when these proteins tend to aggregate spont aneously . For export, the precursor must be maintained in a translocation-competent conformation and the signal peptide must remain exposed to the secretory machinery. Early folding or aggregation of the precursor leads to 2471 loss of translocation. Bacterial chaperone factors, including trigger factor, GroEL, GroES, DnaK and SecB appear to be required to prevent early protein folding and to keep the precursor in a translocation-competent conformation (for review, see Saier et al., 1989; Lee & Olins, 1992). However, the rapid folding of IL-2 in the cytoplasm could not be inhibited by increasing the intracellular amount of GroEL and GroES. Two general problems encountered in the synthesis of eukaryotic proteins in E. coli at a high level are protein aggregation and/or incomplete processing by bacterial peptidase activity. Altering growth conditions has been shown to influence favourably the yield of correctly processed material, e.g. by lowering the temperature (Schein & Noteborn, 1988) or by limited induction (Ghrayeb et al., 1984). Because induction with 1 mMIPTG leads to a high production level of the ssOmpAIL-2 and ssPhoA-IL-2 fusions, we reduced the level of IPTG to 1 0 p in ~ LB and/or decreased the growth temperature to 28 "C. We could not observe any significant difference in the extent of maturation (precursor stability and processing competence) under different conditions (data not shown). Since we could not promote export by the different techniques described above, we tried to apply our experience in the use of lysis proteins to promote nonspecific release of proteins to the extracellular medium. Neither the colicin A lysis protein (Baty et al., 1987a), nor the bacteriophage 4x174 lysis protein E (Witte et al., 1989) promoted the extracellular release of putatively soluble forms of IL-2. IL-2 in its original environment may have some additional factors which influence its expression and secretion, either as directed by its original signal peptide or by other factors such as eukaryotic chaperones. In the case of the maltose-binding protein, attainment of a stable tertiary structure in the cytoplasm correlates with the loss of competence for secretion (Randall & Hardy, 1986).Thus, once a bacterial protein is properly folded in the cytoplasm, it seems that there is no mechanism of repackaging it for export. Binding proteins and chaperones appear to prevent the nascent polypeptide chains from crossing the energy barrier to a folded stable state, either stabilizing the partially folded structure or protecting these incompletely folded, flexible molecules from premature aggregation (Fischer & Schmid, 1990). In a further attempt to promote secretion of IL-2, the mature protein was fused to the C-terminus of the maltose-binding protein. The export of the latter has been studied extensively and plasmid vectors have been designed based on these studies to promote high levels of expression and secretion of recombinant proteins (for review, Bankaitis et al., 1985; Randall et al., 1987). This system allows the protein to fold by itself without Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 03 Aug 2017 19:13:35 2472 G . Halfmann and others interference of an adjacent signal sequence and has been used successfully with various proteins to obtain export in E. coli (Martineau et al., 1992;Bregkgkre & Bedouelle, 1992). This approach was successful and allowed export, processing of the MBP-FX-IL-2 fusion protein and production of active IL-2. Export of preMBP-IL-2 presumably occurs because export begins cotranslationally before completion of the synthesis of the IL-2 moiety. However, we cannot rule out the hypothesis that interaction of the hybrid protein with the SecB chaperone plays a part in preventing misfolding and aggregation. We are grateful to M. Delaage for helpful discussions. We thank J. M. Clement for the anti-maltose-binding protein. We also thank M. Green and H. Rickenberg for careful reading. This work was supported by the Commission of the European Communities, the Ministkre de la Recherche et de la Technologie and the Fondation pour la Recherche Medicale. G. H. was the recipient of an EC postdoctoral fellowship. References BANKAITIS, V. A., RYAN,J. P., RASMUSSEN, B. A. & BASSFORD, P. J., JR (1985). The use of genetic techniques to analyse protein export in Escherichia coli. Current Topics in Membrane Transport 24, 105-1 50. BAR, D., LLOUBES, R., GELI,V., LAZDUNSKI, C. & HOWARD,P. S. (1987a). Extracellular release of colicin A is non-specific. EMBO Journal 6, 2463-2468. BATY,D., KNIBIEHLER, M., VERHEU,H., PATTUS,F., SHIRE, D., BERNADAC, A. & LAZDUNSKI, C. (1987 b). Site-directed mutagenesis of the COOH-terminal region of colicin A: effect on secretion and voltage-dependent channel activity. Proceedings of the National Academy of Sciences of the United States of America 84, 1152-1 156. BENDER, E., KOLLER, K. P. & ENGELS, J. W. (1990). Secretory synthesis of human interleukin-2 by Streptomyces lividans. Gene 86, 227-232. BERNADAC, A. & LAZDUNSKI, C. (1981). Immunoferritin labelling of ultrathin frozen sections of Gram-negative bacterial cells. Biology of the Cell 41, 21 1-216. BETTER, M., CHANG, C. P., ROBINSON, R. R. & HORWITZ, A. H. (1988). Escherichia coli secretion of an active chimeric antibody fragment. Science 240, 1041-1043. BOYD,D. & BECKWITH, J. (1990). The role of charged amino acids in the localization of secreted and membrane proteins. Cell 62, 103 1-1033. B ~ G E G ~ RF.E ,& BEDOUELLE, H. (1992). Expression, export and purification of antibody fragments fused to the maltose-binding protein of Escherichia coli. Compte rendu de PAcademie des Sciences, Paris 314, serie 111, 527-532. DEN~FLE, P., KORVARIK, S . , CIORA,T., GOSSELET, N., BENICHOU, J. C., LATTA,M., GUINET,F., RYTER,A. & MAYAUX,J. F. (1989). Heterologous protein export in Escherichia coli: influence of bacterial signal peptides on the export of human interleukin 1B. Gene 85, 499-5 10. DERMAN,A. I. & BECKWITH, J. (1991). Escherichia coli alkaline phosphatase fails to acquire disulfide bonds when retained in the cytoplasm. Journal of Bacteriology 173, 7719-7722. D~vosR., , PLAETINCK, G., CHEROUTRE, H., SIMONS, G., DEGRAVE, W., TAVERNIER, J., REMAUT, E. & FIERS,W. (1983). Molecular cloning of human interleukin 2 cDNA and its expression in E. coli. Nucleic Acids Research 11, 4307-4323. DUFFAUD, G. D., MARCH,P. E. & INOUYE, M. (1987). Expression and secretion of foreign proteins in Escherichia coli. Methods in Enzymology 153,492-502. FISCHER,G. & SCHMID,F.X. (1990). The mechanism of protein folding. Implications of in vitro refolding models for de novo protein folding and translocation in the cell. Biochemistry 29, 2205-2212. FURSTE,J. P., PANSEGRAU, W., FRANK,R., BLOCKER, H., SCHOLZ, P., BAGDASARIAN, M. & LANKA,E. (1986). Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector. Gene 48, 119-131. GHRAYEB, J., KIMURA, H., TAKAHARA, M., HSIUNG,H., MASUI,Y . & INOUYE, M. (1984). Secretion cloning vectors in Escherichia coli. EMBO Journal 3, 2437-2442. GILLIS,S., FERM, M. M., Ou, W. & SMITH,K. A. (1978). T-cell growth factor : parameters of production and a quantitative microassay for activity. Journal of Immunology 120, 2027-2032. GOLOUBINOFF, P., GATENBY, A. A. & LORIMER,G. H. (1989). GroE heat-shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Escherichia coli. Nature, London 337, 44-47. HATTORI,M. & SAKAKI, Y. (1986). Dideoxy sequencing method using denatured plasmid templates. Analytical Biochemistry 152, 232-238. VON HEIJNE, G. (1986). Net N-C charge imbalance may be important for signal sequence function in bacteria. Journal of Molecular Biology 192, 287-290. JOHNSTON, T. C., THOMPSON, R. B. & BALDWIN, T. 0. (1986). Nucleotide sequence of the IexB gene of Vibrio harveyi and the complete amino acid sequence of the beta subunit of bacterial luciferase. Journal of Biological Chemistry 261, 480548 11. JOSEPH-LIAUZUN, E., LEPLATOIS,P., LEGOUX,R., GUERVENO, V., MARCHESE, E. & FERRARA, P. (1990). Human recombinant interleukin-1 beta isolated from Escherichia coli by simple osmotic shock. Gene 86, 291-295. Ju, G., COLLINS,L., KAFFKA,K. L., TSIEN,W. H., CHIZZONITE, R., CROW,R., BHATT,R. & KILIAN,P. L. (1987). Structure-function analysis of human interleukin-2. Identification of amino acid residues required for biological activity. Journal of Biological Chemistry 262, 5723-573 1. LEE, S . C. & OLINS,P. 0. (1992). Effect of overproduction of heat shock chaperones GroES and DnaK on human procollagenase production in Escherichia coli. Journal of Biological Chemistry 267, 2849-28 52. LLOUB$S, R., BATY,D. & LAZDUNSKI, C. (1986). The promoters of the genes for colicin production, release and immunity in the ColA plasmid : effects of convergent transcription and lexA protein. Nucleic Acids Research 14, 2621-2636. MACINTYRE, S., ESCHBACH, M. L. & MUTSCHER, B. (1990). Export incompatibility of N-terminal basic residues in a mature polypeptide of Escherichia coli can be alleviated by optimising the signal peptide. Molecular and General Genetics 221, 466-474. MARSTON, F. A. 0. (1986). The purification of eukaryotic polypeptides synthesized in Escherichia coli. Biochemical Journal 240, 1-12. MARTINEAU, P., GUILLET, J.-G., LECLERC, C. & HOFNUNG, M. (1992). Expression of heterologous peptides at two permissive sites of the MalE protein : antigenicity and immunogenicity of foreign B-cell and T-cell epitopes. Gene 113, 3546. MESSING,J. (1983). New M13 vectors for cloning. Methods in Enzymology 101, 20-78. MILLER,J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, NY: Cold Spring Harbor Laboratory. NEU, H. C. & HEPPEL,L. A. (1965). The release of enzymes from Escherichia coli by osmotic shock and during formation of spheroplasts. Journal of Biological Chemistry 240, 3685-3692. OKA, T., SAKAMOTO, S., MIYOSHI,K. I., FUWA, T., YODA, K., YAMASAKI, M., TAMURA, S . & MIAYAKE, T. (1985). Synthesis and secretion of human epidermal growth factor by Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 82, 72 12-72 16. PAHWA,R., CHATILA, T., PAHWA,S., PARADISE, C., DAY,N. K., GEHA, N. & GOOD,R. A. (1989). R., SCWARTZ,S . A., SLADE,H., OYAIZU, Recombinant interleukin 2 therapy in severe combined immunodeficiency disease. Proceedings of the National Academy of Sciences of the United States of America 86, 5069-5073. PARKER, K. C. & WILEY,D. C. (1989). Overexpression of native human fl-microglobulin in Escherichia coli and its purification. Gene 83, 117-124. POLLIT, S . & ZALKIN, H. (1983). Role of primary structure and disulfide bond formation in B-lactamase secretion. Journal of Bacteriology 153, 27-32. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 03 Aug 2017 19:13:35 Targeting of IL-2 to periplasm of E. coli RANDALL, L. L. & HARDY,S . J. S . (1986). Correlation of competence for export with lack of tertiary structure of the mature species: a study in vivo of maltose-binding protein in E. coli. Cell 46, 921-928. RANDALL,L. L., HARDY, S. J. S. & THOM,J. R. (1987). Export of protein : a biochemical view. Annual Review of Microbiology 41, 507-541. RENTIER-DELRUE, F., SWENNEN,D. & MARTIAL,J. (1988). PIN-111 ompA secretion vectors: modification of the ompA signal peptide sequence for easier insert cloning. Nucleic Acids Research 16, 8726. ROSENBERG, S. A., SPIESS, P. & LAFRENIERE, R. (1986). A new approach to the adoptive immunotherapy of cancer with tumor-infiltrating lymphocytes. Science 233, 1318-1 321. SAIER,M. H., WERNER,P. K. & MWLLER,M. (1989). Insertion of proteins into bacterial membranes : mechanism, characteristics, and comparisons with the eucaryotic process. Microbiological Reviews 53, 333-366. SCHEIN,C. H. & NOTEBORN, M. H. M. (1988). Formation of soluble proteins in Escherichia coli is favored by lower growth temperature. Biol Technology 6, 29 1-294. SMITH,K. (1988). Interleukin-2 : inception, impact, and implication. Science 240, 1169-1 176. 2473 SUMMERS,R. G . & KNOWLES,J. R. (1989). Illicit secretion of a cytoplasmic protein into the periplasm of Escherichiu coli requires a signal peptide plus a portion of the cognate secreted protein. Journal of Biological Chemistry 264, 2007&2008 1. TANIGUCHI, T., MATSUI,H., FUJITA,T., TAKAOKA, C., KASHIMA, N., YOSHIMOTO, R. & HAMURO, J. (1983). Structure and expression of a cloned cDNA for human interleukin-2. Nature, London 302,305-3 10. TOWBIN,H., STAEHELIN, T. & GORDON,J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets : procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America 76,4350-4354. WATSON,M. E. E. (1984). Compilation of published signal sequences. Nucleic Acids Research 12, 5 145-5 1 64. WITTE,A., BLASI,U., HALFMANN, G., SZOSTAK, M., WANNER,G. & LUBITZ,W. (1989). Phi X174 protein E-mediated lysis of Escherichiu coli. Biochimie 72, 191-200. YAMANE, K. & MIZUSHIMA, S. (1988). Introduction of basic amino acid residues after the signal peptide inhibits protein translocation across the cytoplasmic membrane of Escherichiu coli. Relation to the orientation of membrane proteins. Journal of Biological Chemistry 263, 19690-19696. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 03 Aug 2017 19:13:35