Expression in Escherichia coli of c-type cytochrome genes from Rhodopseudomonas viridis

183

BiJd, imica et Biophysica Acta, 1088 (1991) 183-190 © 1991 Elsevier Science Publishers B.V, 0167-4781/91/$03.50 ADONIS 016747819100075 B BBAEXP 92205

Expression in Escherichia coli of c-type cytochrome genes from Rhodopseudomonas viridis R e i n h a r d G r i s s h a m m e r *, C h r i s t i n e O e c l d a n d H a r t m u t M i c h e l Max-Planck.institut flit Biophysik, Frankfurt / Main, (F.R.G.) (Received 16 July 1990)

Key words: Cytochrome, c-type; Gene expression; Inclusion body; ( Escherichia coil ); ( Rhodopseudomonas viridis )

The genes coOing for the photosynthetic reaction center cytochrome c subunit (pu[C) and the soluble cytochrome c z (cycA) from the purple non-sulfur bacterium Rhodopseudomonas viridis were expressed in Escherichia coll. Biosynthesis of the reaction center cytochrome without a signal peptide resulted in the formation of inclusion bodies in the cytoplasm amounting to 14% of the total cellular protein. A series of plasmids coding for the cytochrome subunit with varying N-terminal signal peptides was constructed in attempts to achieve translncation across the E. coil cytoplasmic membrane and heme attachment. However, the two major recombinant proteins with N-termini corresponding to the signal peptide and the cytochrome were synthesized in E, coil as non-specific aggregates without heine incorporation. An increased ratio of precursor as compared to 'processed' apo-cytochrome was obtained when expression was carried out in a proteinase-deficient strain. Cytnchrome c 2 from R. viridis was synthesized in E. coli as a precursor associated with the cytoplasmic membrane. An expression plasmid was designed encoding the N-terminal part of the 33 kDa precursor protein of the oxygen-evolving complex of Photosystem !i from spinach followed by cytochrome c 2. Two recombinant proteins without heme were found to aggregate as inclusion bodies with N-termini corresponding to the signal peptide and the mature 33 kDa protein.

Introduction The photosynthetic reaction center from the purple non-sulfur bacterium Rhodopseudornonas viridis is a membrane-bound pigment-protein complex that catalyzes the conversion of light energy to chemical energy. Its three-dimensional structure has been elucidated by crystallization and X-ray diffraction analysis (for a review see Ref. 1). After light-driven electron transport across the photosynthetic membrane, electrons are returned to the periplasmic side by the cytochrome bc: complex with accompanying proton translocation and

Abbreviations: IPTG, isopropyl-fl-o-thiogalactopyranoside; kDa, kilodahon; RC, reaction center; R. viridis, Rhodopseudomonas v#idis; SDS, sodium dodecylsulfate; SDS-PAGE, sodium dodecylsulfatepolyacrylamide gel electrophoresis; Tween 20, poly(oxyethylene)sorbitan monolaurate. *Present address: 1RC for Protein Engineering, MRC, Cambridge, U.K. Correspondence: R. Grisshammer, Max-Planck-lnstitut fiir Biophysik, Heinrich-Hoffmann-Strasse 7, D-6000 Frankfurt/Main, F.R.G.

energy conservation. Re-reduction of the photooxidized primary electron donor of the reaction center is accomplished via the soluble cytochrome c2 [2] and another cytochrome which is an integral component of the reaction center from R. viridis. Little is known about the interaction between cytochrome c2 and the reaction center cytochrome, as well as the amino acid residues of the cytochrome subunit participating in electron transfer to the primary electron donor. Site-specific mutagenesis is one possibility to investigate in detail the role of individual amino acid residues in protein-protein interaction and electron transfer. Heterologous expression of the R. viridis cytochrome genes in, e.g., E. coil and subsequent in vitro experiments were considered as one approach to address this topic by means of site-specific mutagenesis. In this paper we report the expression in E. coli of the reaction center cytochrome (pufC) [3] and cytochrome c2 (cycA) [4] genes from R. viridis and present a detailed analysis of the expression products. A series of plasmids was constructed for the expression of the cytochrome subunit and the soluble cytochrome c2, either with or without signal sequences needed for the translocation of the cytochromes across the cytoplasmic

184 membrane. In addition, cytochrome c2 was synthesized as a fusion protein with the N-terminal part of the 33 kDa protein of the oxygen evolving complex of Photo~ystem I1 from spinach. Materials and Methods

Bacterial strains and growth conditions. Escherichia cob strain DH5a (Bethesda Research Laboratories, Neu Isenburg, F.R.G.) 151 was used as the host for recombinant plasmids and grown in LB-medium [5] or on LB agar plates containing the appropriate antibiotics (ampiciilin 100 ~tg/ml and kanamycin 50 pg/ml). The medium was supplemented with 0.5% or 1% KNO 3, 0.3% trimethylamine-N-oxide (TMAO), or 0.3% dimethyl sulfoxide (DMSO) as external electron acceptors, and optionally with 1% glucose for expression experiments under anaerobic growth conditions. Expression of recombinant cytochrome genes was performed in E. coli strains DH5a and CAG 629 (SC122 Ion - htpR- ) [61. Genetic manipulations. Restriction endonucleases and DNA-modifying enzymes were obtained from Boehringer (Mannheim, F.R.G.) or Bethesda Research Laboratories (Neu lsenburg, F.R.G.). Cloning and other molecular biological methods were used as described [5,7]. Oiigodeoxynucleotides, used for plasmid constructions, were synthesized on an Applied Biosystems 380 B and kindly provided by D. Oesterhelt, Martinsried, F.R.G. Total RNA from E. coli was prepared as described in Ref. 7 from 10 ml cultures. Northern blot analysis and synthesis of radioactively labeled hybridization probes were performed as in Ref. 4. Expression plasmid~. The expression plasmid pDS12/ RBSll [8] contains the strong coliphage T5 promoter PN25 fused with the lac operator. Transcription therefore is controlled by the Lac repressor, which is overproduced by the compatible plasmid pDMI, 1 [9], present in all E. coli strains used for expression with pDS-vectors. Transcription of foreign genes in plasmid plN-III-A 110] is started from a tandem promoter consisting of the constitutive ipp-promoter followed by the inducible lac-promoter/operator element. The expression plasmids and their characteristics used in this study are listed in Table I. Parts of the amino acid sequences of the reaction center (RC) cytochrome [3] and cytochrome c 2 [41 as deduced from the DNA sequences of the genes are shown for comparison in this table. Plasmids piN-Ill-31 and piN-Ill-38 were constructed for synthesis of a shortened (31 kDa) and full-length (38 kDa) RC cytochrome without a signal peptide. For translocation across the cytoplasmic membrane, plasmids were designed encoding the RC cytochrome with either an N-terminal enterotoxin B signal

peptide of E. coil (pDS-SE31, pDS-SE38) or the leader peptide of the RC cytochrome (pDS-SV38). Similarly, the vectors pDS-SVC2 and pDS-SEC2 were constructed for translocation of cytochrome c 2 into the periplasm of E. coli and heme attachment, using the signal peptides of cytochrome c 2 or enterotoxin B. Plasmid pDS1233-C2 was designed to code for a fusion protein consisting of a signal peptide, the N-terminal 153 amino acids of the 33 kDa protein of the oxygenevolving complex of Photosystem 1I from spinach [11,12] followed by cytochrome c 2. Protein-chemical methods. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Laemmli [13]. Gels were stained for the presence of hemes by measuring peroxidase activity of the heme groups according to Thomas et al. [14]. In order to estimate the relative amounts of electrophoreticaily separated proteins, Coomassie-bluestained SDS-gels were scanned using a Hoefer GS- 300 scanning densitometer. Determination of the total amount of protein was performed according to a modified Lowry procedure in the presence of SDS [15]. For N-terminal sequencing, recombinant proteins were electrophoretically transferred onto poly(vinylidene) difluoride membranes (lmmobilon, Millipore) [16] after SDS-PAGE. The transfer buffer consisted of 25 mM Tris-acetate (pH 8.3), 0.1% SDS, 0.5 mM dithiothreitol, 10% methanol. After staining with Amido black (Serva), analysis of the respective peptides was carried out on an Applied Biosystems 477A Peptide Sequencer by E. Kojro and F. Fahrenholz. Inclusion bodies were prepared as described [17]. All steps were performed at 4" C. E. coli cells were harvested by centrifugation and lysed for 30 min in the presence of lysozyme (2 mg lysozyme/ml in 50 mM Trs-HCI (pH 8.0), 25% (w/v) sucrose, 1 mM EDTA). Chromosomal DNA was degraded with DNase I (Sigma) (10 # g / m l ) in the presence of 10 mM MgCI 2 and 1 mM MnCI2 for 30 min. After addition of detergent-containing buffer (200 mM NaCI, 1% (w/v) deoxycholate, 1% (w/v) Triton X-100, 20 mM Tris-HC! (pH 7.5), 2 mM EDTA, 10 mM fl-mercaptoethanol), the insoluble proteins were pelleted at 7000 x g for 10 min and washed with Triton X-100 buffer (0.5% (w/v) Triton X-100, 1 mM EDTA, 20 mM Tris-HCl (pH 7.5), 10 mM flomercaptoethanol). For analysis of the subceUular localization of recombinant proteins in E. coli, cells were harvested, washed and resuspended in shock buffer (20 mM Tris-HCl (pH 8.0), 2 mM EDTA, 20% (w/v) sucrose). After incubation for 10 min at room temperature and subsequent centrifugation (16000 x g, 2 min) the cells were resuspended in cold water. The periplasmic fraction (supernatant) was separated from the spheroplasts (pellet) by centrifugation (16 000 x g, 2 rain). The spheroplasts were incubated in lysozyme-containing buffer (100 #g lyso-

185 z y m e / m l in 50 mM Tris-HCl (pH 8.0). 20 mM EDTA) for 15 min at 3 7 ° C and broken by repeated freeze-thaw cycles. Inclusion bodies were recovered by centrifugation at 7000 x g (10 rain) after DNase I treatment (10 # g / m l ) in the presence of 10 m M MgC! 2 and ! ram MnC! 2 for 30 min at 4 ° C . Membranes were isolated from the supernatant by ultracentrifugation (250000 x g, 30 min, 4 ° C ) (181. Western blot analysis. Electrophoretic transfer of proteins [19] to Immobilon membranes was performed with a Semi-dry Biometra Fast Blot apparatus. Transfer conditions were as recommended by the manufacturer. Recombinant R. viridis cytochromes synthesized in E. coli were detected as follows: TBST-buffer (10 mM Tris-HCI (pH 8.0), 150 m M NaCI, 0.05% ( w / v ) Tween 20, 0.02% ( w / v ) NAN3) was used for all procedures. The Immobilon membrane was blocked with 1% bovine serum albumin in TBST-buffer and incubated for 1 h with anti-cytochrome anti-serum (see below). Washing between incubation steps was done with TBST-buffer. The Western blot was developed by using goat anti-rabbit IgG conjugated to alkaline phosphatase with the substrates 5-bromo-4-chloro-3-indolyl phosphate and Nitro blue tetrazolium. S e r u m preparation. T h e R C cytochrome subuait from R. oiridis was purified as described previously [3]. Cytochrome c 2 from R. uiridis was prepared according to Weite et aL [20]. Freund's complete adjuvant and the respective cytochromes were mixed and emulsified, injection into rabbits, boosting, and serum preparation have been described [21].

Results and Discussion

Synthesis o f the reaction center cvtochrome in the absence o f a signal peptide

The cytochrome c subunit is the largest subunit in the RC complex and is bound to a flat surface of the L and M subunit at the periplasmic side of the membrane. It consists of an N-terminal segment of approx. 55 amino acid residues, two pairs of heme-binding segments (hemes 1 + 2 and hemes 3 + 4), and a segment connecting the two pairs. Each heme-binding segment consists of a helix followed by a turn and the hemebinding sequence -Cys-X-Y-Cys-His-. The hemes are connected to the cysteine residues via thioether linkages. The two pairs of heme-binding segments are related by a local two-fold symmetry [1,22]. The cytochrome subunit has surface properties typical of watersoluble proteins. Nevertheless, it displays membraneprotein-like qualities and aggregates easily due to a lipid-type membrane anchor [23]. The N-terminal cysteine of the mature cytochrome is linked to a glycerol residue via a thioether bridge; two fatty acids are esterified to the hydroxyl groups of the glycerol residue. Two plasmids piN-Ill-31 and p l N - l l l - 3 8 were constructed in order to obtain expression of a soluble cytochrome subunit. Plasmid plN-111-31 (Table I) contains the gene coding for a shortened cytochrome subunit (31 kDa) without the first 55 amino acids whi,,h form a long connection between the first heme attachment site and the diglyceride membrane anchor. The

TABLE ! Plasmids used for expression in this stua~v.

PlasmJd plN-III-A pDSI2/RBSII pDMI, l Plasmid

Ref. 10 8 9 Recombinant cytochrome

RC eytochrome from R. viridis piN-Ill-31 RC cytochror,.le piN-Ill-38 RC cytochro~e pDS-SE31 RC cytochr~,me pDS-SE38 RC cytochrome pD~SV38 RC cytoch,,ome Cytochrome c2 from R. viridis pDS-SEC2 cytochrome c2 pDS-SVC2 cytochrome c2 pDS1233-C2

Characteristics Apr, lacL Apr, Kmr, laclq.

Promoter lppP/lac p° T5 Pw25/lac°

col E1 replicon col El replicon pAl5 replicon

Signal peptide NKGLIVNSVATVALASLVAG;CIFE..YS41( MSY~KNV

enterotoxin B enterotoxin B R. viridis RC cytochrome enterotoxin B R. viridis c2

33 kDa protein of the oxygen-evolving complex of Photosystem II with cyt. c2

MAF z E NNKVKCYVLFTALLSSLYAHGzSysaI(NV RNKVKCYVLFTALLSSLYAHG MRXLZVNSVATVALASLVA6

SE 3 C1

NRKLVFGLFVLAASVAPAAAZGtDAA MNKVKCYVLFTALLSSLYAHGtSZPAAa I RRKLVFGLFVLAASVAPAAA ....

RGSls3ZPAAG 1

G5

N V . • K s3¢; • . . . . g 336 .... g $36 ....

g $36

....

....

K 3-~' g $34

.... ....

K 1°? Ktot

....

K ~°7

....

Kt o t

• Numbers refer to amino acid positions in the R. viridis RC eytochrome [3l, ¢ytochrome c2 [4l, or the 33 kDa protein of the oxygen evolving complex of Pl~otosystem II [11,12l. Bold letters indicate authentic residues of the RC ¢ytochrome or cytochrome c2 from R. viri~s. Arrows: potential cleavage sites for signal peptidases.

186 vector piN-Ill-38 (Table I) encodes a full length RC cytochrc,me (38 kDa) with the altered amino-terminus Met-Ala-Phe- instead of Cys-Phe-. E. coil DH5a cells containing the plasmid piN-Ill-31 were grown aerobically at 37°C and induced with 1 mM IPTG in the mid-exponential phase. An abundant expression product of approx. 31 kDa, corresponding to the expected molecular mass, was detected by analyzing extracts of whole cells by SDS-PAGE (Fig. 1A, lane 2). The recombinant 31 kDa apo-cytochrome without heine was isolated as a pellet by a low-spin centrifugation step after lysing the cells (Fig. 1A, lane 5). It could be solubilized only with strong denaturants such as urea, SDS or guanidinium-HCI, indicating that the 31 kDa apo-cytochrome accumulated in E. coil in the form of inclusion bodies [24]. Sequence analysis (Ser-Tyr-LysAsn-Val-) revealed an N-terminus of the recombinant 31 kDa apo-cytochrome correctly processed by deformylase and methionine-aminopeptidase. Maximal yields of up to 30 mg per i culture corresponding to 14% of the total cellular protein were obtained after 2 h of induction (data not shown). Western blot analysis using an anti RC-cytochrome anti-serum displayed synthesis of the 31 kDa apo-cytochrome also in the absence of IPTG (Fig. 1B, lane 3) as insoluble aggregates due to incomplete repression of the transcription of the cytochrome gene. This phenomenon had been observed earlier [25] with expression experi-

ments performed in rich medium using the piN-Illplasmid family. The formation of inclusion bodies is not only limited to "foreign' proteins that are synthesized in E. coli, but was observed as well for normal E. coli proteins produced at high levels using recombinant DNA techniques (for a review see Ref. 24). One reason for inclusion body formation might be a high local concentration of the recombinant protein in the cytoplasm leading to non-specific precipitation [26]. Therefore, expression in E. coli DH5a/plN-III-31 was performed at 22°C instead of 370C. However, no soluble 31 kDa apo-cytochrome was detected in E. coil (in addition to the inclusion bodies) with or without induction. This indicates that even at a reduced production rate, no direct correlation exists between the amount of insoluble recombinant protein and the protein concentration. The reasons for inclusion body formation are poorly understood [24]. In the case of the recombinant 31 kDa apo-cytochrome, correct folding might be impaired without covalent heine attachment, thus allowing intermolecular contacts between domains not exposed on the surface of the mature RC cytochrome and subsequent aggregation. No expression was detected in E. coli harboring plasmid piN-Ill-38 (Table 1), although transcription of the cytochrome gene was not impaired as shown by Northern blot analysis (data not shown). Reduced el-

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Fig. l. Synthesisof the shortened 31 kDa RC apo-cytochromewithout a signal peptide (plN-lll-3l). E. coli DH5a cells harboringplasmid plN-lll-3l were grownaerobicallyin LB-mediumand inducedwith 1 mM IPTG for 2 h. (A) Sampleswere analyzedby 12~ SDS-PAGEand protein bands were stainedwith Coomassieblue.(B) CorrespondingWesternblot analysis.Lane 1: lysedcells with parental plasmidpIN-Ill-A, induced; lanes2 and 3: lysedcells with plasmidpiN-Ill-31,inducedand non-induced,respectively;lanes4 and 5: supernatantand pellet of the inclusionbodypreparationof inducedcellscontainingpiN-Ill-31,respectively;lane M: molecularweightmarkers.

1~7

ficiency of translation initiation and subsequent proteolytic degradation of the recombinant cytocbrome might account for the observed effect. Efficient translation initiation is not only dependent on a Shine-Dalgarno sequence [271 (which is identical in the plasrr,ids piN!11-38 and p i N - I l l - 3 1 ) in the correct distance from the start codon, but also on free accessability of the AUG codon in the transcript. Therefore, possible secondary structures of the cytochrome transcripts were analyzed with the computer program F O L D [28]. In the case of plN-llI-38, the A U G start codon of the cytochrome was located in a stem of a potential stem-loop structure. This might reduce the efficiency of translation initiation. In the case of piN-Ill-31, the start codon was found in a loop region. Synthesis of the reaction center cytochrome with an Nterminal signal peptide When grown anaerobically in the presence of nitrate, a soluble c-type cytochrome :352, which is located in the periplasmic space, is synthesized in E. coil [291. The c-type cytochromes from R. viridis are found at the peripiasmic side of the photosynthetic membrane and are synthesized as precursor proteins with an N-terminal signal peptide as deduced from the DNA-sequence of the corresponding genes [3,4,301. Consequently, expression plasmids were constructed encoding the RC cytochrome with an N-terminal signal peptide for translocation across the E. coil cytoplasmic membrane. Expression was performed under anaerobic conditions in LB medium supplemented with the external electron acceptor nitrate, trimethylamine-N-oxide (with or without glucose) or dimethyl sulfoxide. Under these conditions endogenous c-type cytochromes from E. coil were synthesized as shown by SDS-PAGE and measurement of the peroxidase activity of the heme groups (see also Ref. 31). Piasmid pDS-SE31 is predicted to express a 34 kDa protein (Table I), consisting of the enterotoxin B leader peptide and the shortened 31 kDa cytochrome. Induction of cells containing pDS-SE31 resulted in synthesis of two major recombinant proteins identified by Western blot analysis (Fig, 2). The recombinant cytochromes were heme stain negative and found to precipitate as inclusion bodies irrespective of the duration of induction and the composition of the growth media. N-terminal sequence analysis of the larger 34 kDa protein revealed the sequence Met-Asn-Lys-Val-Lys- of the enterotoxin B signal peptide, indicating that the formylmoiety of the initiating formyl-methionine was removed by a deformylase in E. coil The smaller expression product displayed the sequence X-Tyr-Lys-Asn-Val- of the shortened 31 kDa apo-cytochrome without the signal peptide (Table I). Expression studies were performed in E. coil DH5a as well as in the proteinase deficient strain C A G 629 [6]. A shift towards the 34

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Fig. 2. Synthesis of the 34 k D a RC pr¢-apo--cytochrome wtth an N-terminal entero~oxin B signal pepttde (pDS--SE31~. E. cob cells were grown anaerobically in LB-medium st:pplemented with nitrate at 30°C and induced for 1 h with IPTG. Lysed cells were analyzed by 12~ SDS-PAGE and subsequent immuno-blotting. Lane 1: E cob strain D H S a with parental plasmid p D S I 2 / R B S i l , induced with 1 mM IPTG; lane 2: D H S a with pDS-SE31, non-induced; lane 3: E. colt strain C A G 629 with pDS-SE31, induced with 20 #M IPTG: lane 4: D H 5 a with pDS-SE31, induced with 20 tiM IPTG; l a n ~ 5. 6 and 7,8 as lanes 3, 4. but induced with 60 #M IPTC and 1 mM IIrlG, respectively. Lanes 9 and 10: D H S a h a r b o u n n g piN-ill-21, non-induced and induced with 1 mM IPTG, Arrows indicate the position of the 34 kDa precursor and 31 kDa RC apo--cytochrome.

kDa pre-apo-cytochrome was detected in the protease deficient strain in comparison to DH5a (Fig. 2. lanes 3-8). There are two possible interpretatinns of these results: (1) partial export ef the recombinant 34 kDa pre-apo-cytochrome across the membrane, removal of the signal peptide by signal peptidase I and subsequent aggregation of the 31 kDa apo-cytochrome in the periplasm or (Ii) preferential partial proteolytic degradation at the site between the signal peptide and the mature protein by proteinases in the cytoplasm. The latter possibility is considered likely, because more 34 kDa pre-apo-cytochrome was obtained using a proteinase deficient E. coil strain for expression in comparison to DH5a (Fig. 2). However. it is surprising that the same substrate specificity is displayed by signal peptidase I and the cytoplasmic proteinase(s). The plasmid pDS-SE31 previously discussed codes for a shortened RC cytochrome missing the N-terminal 55 amino acids of the mature R. viridis cytochrome subunit. H,:,wever, part of the information for heme attachment might be contained in the N-terminus of the RC cytochrome. Therefore, plasmids pDS-SE38 and pDS-SV38 (Table I) were designed for synthesis of a 40 kDa precursor cytochrome containing the enterotoxin B signal sequence or the R. viridis RC cytochrome signal peptide and the full length cytochrome. The enterotoxin B leader peptide of the precursor cytochrome encoded by plasmid pDS-SE38 v,as expected to be removed by signal peptidase I. The cytochrome encoded by plasmid pDS-SV38 should be cleaved by signal peptidase ii; the

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Fig. 3. Expression of RC cytochromes from the plasmids pDS-SE38 and pDS-SV38. E. coil DHSa cells were grown anaerobically in LB-medium supplemented with nitrate at 25 °C and induced with 100 v-M IPTG for 2 h. Samples were analyzed by 12~ SDS-PAGE and subsequent immuno-blotting. Lane 1: lysed cells containing the parental plasmid pDSI2/RBSll, induced; lanes 2 and 3: pDS-SE38, lysed cells, induced and non-induced; lanes 4 and 5, supernatant and pellet of the inclusion body preparation of induced cells containing pDSSE38; lanes 6-9: as lanes 2-5. using E. coli DHSa containing plasmid pDS-SV38.

N-terminal cysteine residue of the mature cytochrome might be additionally modified similarly to that in R. viridis [23] or to the major outer membrane lipoprotein of E. coil [32,33]. Both recombinant proteins aggregated as inclusion bodies (Fig. 3) and showed no heine stain activity (data not shown). Furthermore, several expression products of decreasing molecular mass, as detected by Western blot analysis, indicated proteolytic degradation of the 40 kDa pre-apo-cytochromes when analyzing whole cell extracts. The proteolytic pattern changed after preparation of the inclusion bodies by low-speed centrifugation and subsequent Triton X-100 washes, probably due to increased proteinase activity after cell disruption. The time period of induction and the composition of the growth media had little effect on the above described results. Therefore, the use of different signal peptides and varying expression conditions seemed to exert little influence on the observed mode of recombinant cytochrome production in E. coli. Expression of the cycA gene encoding cytochrome c 2 Expression in E. coil of the gene coding for the soluble cytochrome c 2 from R. viridis was performed under anaerobic growth conditions with the external electron acceptor nitrate or trimethylamine-N-oxide. The plasmids pDS-SEC2 and pDS-SVC2 (Table I) direc the synthesis of precursor cytochromes c 2 with the signal peptides of enterotoxin B or cytochrome c 2, respectively. Similar results were obtained with both expression vectors, independent of the growth medium c o r n -

position. After 1 to 2 h of induction, the expression products were found in the supernatant when using the inclusion body preparation method (Fig. 4, lane A). Because this supernatant contains cytoplasmic and periplasmic proteins as well as membranes, subcellular localization analysis was performed. The cytochrome c 2 precursor was associated with the membrane fraction (data not shown), with slightly increased molecular mass in comparison to the mature cytochrome c 2 purified from R. viridis and did not exhibit peroxidase activity. An immediate stop in growth was observed after induction of D H S a cells harboring pDS-SEC2 or pDSSVC2. Prolonged induction (overnight) resulted in cell lysis. This toxic effect might indicate blocked export machinery because of the overproduced pre-apo-cytochrome c 2. In contrast, E. coli cells containing expression plasmids coding for R C cytochromes did not exhibit these effects upon induction. Recently, heterologous expression in E. coli of the cytochrome c 2 genes from Rhodobacter sphaeroides [31] and Rhodospirillum rubrum [34] has been reported. Recombinant holo-cytochrome c 2 was found to be localized in the periplasm in low amounts. Here, expression of plasmids pDS-SVC2 and pDS-SEC2 (Table I) coding for R. viridis cytochrome c 2 yielded membrane-associated pre-apo-cytochrome c 2 with the respective signal

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Fig. 4. Expression of the cytochrome c2 gene (pDS-SVC2). E. coil DHS,~ cells were grown anaerobically in LB-medium supplemented with nitrate at 30°C. Samples of whole cells, induced with 300 pM iPTG for 2 h, were analyzed by the Western blot technique. Lane 1: purified holocytochi,,me c2 from P~ vo'idis; lane 2: lysed DHSa cells containing the parental plasmid pDSI2/RBSII, induced; lanes 3 and 4: pellet and supernatant with the membrane fraction of the inclusion body preparation of induced cells harbouring pDS-SVC2; lanes 5 and 6: lysed cells containing pDS-SVC2, with and without induction. Arrows indicate the position of the recombinant pre-apo-cytochrome c2 and the mature holocytochromec2, respectively.

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the mature 33 kDa protein, thus resembling the expres-. sion pattern of plasmid pDS-SE31. Covalent heine attachment is a prerequisite for functional synthesis of c-type cytochromes from R. viridis in E. coll. It is possible that E. coli cytochrome c heine lyases do not have the proper specificity to process the R. viridis c-type pre-apo-cytochromes. Consequently, heme attachment and translocation across the membrane cannot occur. In addition, chaperone-like proteins [40,41], probably present in R . , iridis and preventing aggregalton of the R C cytochrome precursor may be missing in /L coil Therefore, further experiments are necessary, to understand bacterial c-type cytochrome biogenesis. Acknowledgements

Fig. 5. Synthesis of apo-cytochrome c2 as a fusion protein (pDS1233C2). E. coil DHSa cells were grown anaerobically at 30°C with nitrate as an external electron acceptor and induced for 1 h. Samples were analyzed by immuno-hlottmg. Subcellular localization was performed as described in Materials and Methods. Lane 1: membranes, lane 2: cytoplasmic fraction, lane 3: low-spin centrifugation pellet, lane 4: periplasmic fraction of cells harbouring pDS1233-C2, induced with 300 ~tM IPTG. Lanes 5 and 6: whole cell extract of uninduced and induced cells containing pDS1233-C2. Arrows indicate the position of the fusion protein with and without signal peptide. peptide (Fig. 4). Studies on the biogenesis of Neurospora crassa cytochrome c t [35,36] have revealed that covalent heme attachment occurs at the 'periplasmic' side of the inner mitochondrial membrane catalyzed by cytochrome c t berne lyase, followed by the proteolytic removal of the signal peptide [36]. Despite being coded for by a nuclear gene, cytochrome c~ biogenesis is considered to be similar to bacterial cytochrome biogenesis according to the hypothesis of the endosymbiontic origin of mitochondria and conservative intramitochondrial sorting [37-39]. If the cytochrome biosynthesis is indeed similar, the removal of the signal peptide of the recombinant pre-apo-cytochrome c2 cannot occur due to lack of heme incorporation. In order to achieve translocation across the cytoplasmic membrane of E. coil, an expression plasmid was designed to take advantage of a functional export system. The 33 kDa protein from the oxygen evolving complex of photosystem II from spinach was synthesized in E. coil and secreted into the periplasm [11]. Therefore, the cycA gene was fused to the D N A coding for the signal peptide and the N-terminal 153 amino acids of the 33 k D a protein. Expression of the plasmid pDS1233-C2 (Table I) yielded two products that could be isolated by low-spin centrifugation (Fig. 5, lane 3). Both showed no peroxidase activity, indicating that the heine groups were not attached. Sequence analysis of the smaller protein revealed the N-terminal residues of

We thank Professor D. Oestcrhelt for providing the oligonucleotides, Dr. E. Kojro and Dr. F. Fahrenholz for the N-terminal sequence analysis of the peptides and Dr. H, Koepsell for advice in antibody production. A. Seidler and Dr. E.G. McCarthy provided plasmid pDS1233 and E. coli strain C A G 629. The p D S 1 2 / RBSII-vectors were a gift from Prof. H. Bujard; plasmid p l N - I I I - A was obtained from Dr, M. Inouye. Financial support was obtained from the Deutsche Forschungsgemeinschaft (Leibniz Programm), the MaxPlanck-Gesellschaft and the Fonds der Chemischen Industrie. References

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