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Expression of recombinant Pseudomonas stutzeri di-heme cytochrome c4 by high-cell-density fed-batch cultivation of Pseudomonas putida
Protein Expression and Purification 27 (2003) 175–181 www.elsevier.com/locate/yprep
Expression of recombinant Pseudomonas stutzeri di-heme cytochrome c4 by high-cell-density fed-batch cultivation of Pseudomonas putida Marianne H. Thuesen, Allan Nørgaard, Anne M. Hansen, Mikael B. Caspersen, and Hans E.M. Christensen* Department of Chemistry, Building 207, Technical University of Denmark, 2800 Lyngby, Denmark Received 16 July 2002, and in revised form 20 August 2002
Abstract The gene of the di-heme protein cytochrome c4 from Pseudomonas stutzeri was expressed in Pseudomonas putida. High-yield expression of the protein was achieved by high-cell-density fed-batch cultivation using an exponential glucose feeding strategy. The recombinant cytochrome c4 protein was purified to apparent homogeneity and analyzed by electronic absorption spectroscopy, nanoflow electrospray ionization time-of-flight mass spectrometry, and electrochemistry. Cyclic voltammograms and UV–vis electronic absorption spectra were indistinguishable from the equivalent data of native P. stutzeri cytochrome c4 . Furthermore, the calculated and experimentally determined molecular masses of recombinant cytochrome c4 were identical. Biochemical characterization of both wild-type and mutant derivatives of the protein will be greatly enhanced and facilitated by the described high-yield fermentation and rapid isolation procedure. Ó 2002 Elsevier Science (USA). All rights reserved.
Cytochrome c4 is a 20 kDa bacterial di-heme electron transfer protein [1]. As revealed by X-ray crystal structures, the protein consists of two similarly folded domains, each containing a heme group [2,3]. Two-center metalloproteins are the simplest representative of multicenter metalloproteins and can thus serve as a model system for studying cooperative features in electron transfer proteins with a more complicated kinetic scheme [1]. In this context, studies of site-directed mutants are expected to give important information on the relationship between structure and cooperative behavior. Besides cytochrome c4 , only a few other twocenter metalloproteins are known, e.g., cytochrome cd1 nitrite reductase [4], cytochrome c peroxidase [5], the copper containing nitrite reductase [4], and certain ferredoxins [6]. Cytochrome c4 belongs to the class of c-type cytochromes [7] in which the heme groups are covalently *
Corresponding author. Fax: +45-45-88-31-36. E-mail address: [email protected] (H.E.M. Christensen). URL: http://www.kemi.dtu.dk.
attached to the protein via bonds to two cysteines. The covalent attachment of the heme group is believed to be the cause of the variable success of expression of c-type cytochromes in Escherichia coli as compared to other heme proteins [8]. However, the problem of expression of c-type cytochromes in E. coli has recently been overcome by coexpression of either yeast cytochrome c heme lyase [8] or of the eight cytochrome c maturation genes, the so-called ccm genes [9–11]. Another approach for expression of a c-type cytochrome (Pseudomonas aeruginosa cytochrome c-551) is based on the combination of the pNM185 expression plasmid and the Pseudomonas putida PaW340 strain as an expression host [12]. Several cytochrome c4 genes have been cloned and sequenced [1] including the gene encoding the Pseudomonas stutzeri cytochrome c4 [13], but an expression system for production of recombinant cytochrome c4 has so far not been reported. The subject of high-cell-density cultivation of microorganisms was reviewed recently [14,15]. The aim of the technique is to increase the production of the compound of interest per unit volume. In a study of the production
1046-5928/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 4 6 - 5 9 2 8 ( 0 2 ) 0 0 5 7 5 - 2
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of poly(3-hydroxyalkanoates), it was shown that P. putida grows efficiently to high-cell-density on glucose as a sole carbon source [16], but there has been no report on high-cell-density cultivation of P. putida for recombinant protein production. Here, we report on expression of recombinant P. stutzeri cytochrome c4 by high-cell-density fed-batch cultivation of P. putida, purification of the recombinant cytochrome c4; and its initial characterization by a number of chemical and physical techniques.
Materials and methods Synthetic oligonucleotides, LB media, Yeast Extract, and Peptone 140 were obtained from Life Technologies. Glucose (G5400) and d-aminolevulinic acid–HCl (A3785) were obtained from Sigma–Aldrich. An m-toluate stock solution with a nominal concentration of 100 mM was prepared from m-toluic acid (Sigma–Aldrich, T3,660-9) by first grinding the chemical in a mortar with a pestle and then suspending it in 1 M NaOH, followed by sonication for an extended period of time. Subsequently, the pH was adjusted to 7.5 with 1 M HCl. The resulting yellowish milky suspension was filtered through a 0:45 lm HPLC buffer filtration system and finally sterilized using a 0:2 lm sterile filter. All other chemicals were of analytical grade and solutions were prepared using 18:2 MX cm QPAK Milli-Q water (Millipore). Construction of cytochrome c4 expression vector The P. stutzeri cytochrome c4 gene has previously been cloned on a 3.5-kbp DNA fragment [13]. The original EcoRI restriction enzyme cleavage site was maintained and a BamHI and a SacI restriction enzyme site were introduced after nucleotide number 696 by the use of PCR. Using the protocol provided by the manufacturer, the 0.7kbp gene sequence was amplified with Pyrococcus furiosus DNA polymerase (Stratagene) and cloned into the pPCRScript Amp SK vector (Stratagene). The DNA sequence was confirmed using a BigDye terminator chemistry protocol (Applied Biosystems) and analyzed on an ABI310 (Applied Biosystems) DNA sequencing instrument. The cytochrome c4 encoding EcoRI/SacI DNA fragment was ligated with the EcoRI/SacI restriction enzyme digested pNM185 expression plasmid [17]. Pseudomonas Putida PaW340 cells [18] (DSM, strain No. 2112) were transformed with the resulting cytochrome c4 expression plasmid by electroporation essentially as described in [19,20]. The PaW340 strain was cultured in LB medium supplemented with 1000 mgL1 streptomycin at 30 °C. Inactivation of the streptomycin resistance gene on the pNM185 plasmid was accomplished by insertion of the cytochrome c4 gene. For selection of the pNM
185 plasmid, the LB media were supplemented with 100 mgL1 kanamycin. High-cell-density cultivation procedure A Biostat B fermentor with a 5 L autoclavable jacketed glass vessel (B. Braun Biotech International) equipped with a Mettler Toledo dissolved dioxygen sensor and a Mettler Toledo gel combination pH electrode was used. Prior to autoclaving, the pH electrode was calibrated. During cultivation, the temperature was maintained at 30 °C and the pH at 6:75 0:1 by PIDcontrolled addition of 6 M ammonium hydroxide. Adjustment of the zero-point and the 100% setting of the dioxygen electrode were performed after flushing the fermentor with dinitrogen and with a 5 L/min flow rate of air at a stirrer speed of 800, respectively. The level of dissolved dioxygen was maintained at 40% by using a double cascade in which the stirrer speed was first increased to a maximum of 800 rpm, followed by a gasmix-mode where the airflow was enriched with pure dioxygen to maintain the required level of dissolved dioxygen in the medium. The gas flow was maintained at 5 L/min. Antifoam 289 (Sigma–Aldrich, A5551) was added to a concentration of 20 ppm at the onset of the fermentation and then automatically added as required throughout the fermentation. The semi-synthetic 2.5 L initial batch medium contained (a) 32:5 g KH2 PO4 , 10 g ðNH4 Þ2 HPO4 , 5 g NH4 SO4 , 10 g yeast extract, 4.25 g citric acid, and 50 ml trace element stock solution [21]; (b) 3:0 g MgSO4 7H2 O; (c) 50 g D (+)-glucose; (d) 0.25 g thiamine–HCl; (e) 1 mM m-toluate; (f) 0.1 mM d-aminolevulinic acid; and (g) 100 mgL1 kanamycin. Components (a)–(c) were autoclaved separately, while components (d)–(g) were added as filter sterilized solutions. The recipe of the medium is partly based on the work of Shin et al. [21]. Feed medium I (1 L) contained (a) 800 g D (+)-glucose; (b) 2:0 g MgSO4 7H2 O; (c) 2:0 g ðNH4 Þ2 SO4 ; (d) 20 ml trace element stock solution [21]; (e) 1 mM mtoluate; (f) 0.1 mM d-aminolevulinic acid; and (g) 100 mgL1 kanamycin. Components (a)–(c) were autoclaved separately, while components (d)–(g) were added as filter sterilized solutions. Feed medium II (0.5 L) contained (a) 50 g yeast extract; (b) 100 g peptone 140; (c) 0:5 g MgSO4 7H2 O; (d) 1 mM m-toluate; (e) 0.1 mM d-aminolevulinic acid; and (f) 100 mgL1 kanamycin. Components (a)–(b) were autoclaved separately, while components (c)–(f) were added as filter sterilized solutions. Pre-cultures were grown overnight from )80 °C glycerol stocks on Luria–Bertini (LB) medium agar plates supplemented with 100 mgL1 kanamycin at 30 °C. A single colony was used to inoculate 3 mL liquid LB medium supplemented with 100 mgL1 kanamycin grown at 250 rpm, 30 °C to an OD of 0.6–1.0, and kept
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overnight at +4 °C. Then 0.5 mL was used to inoculate 50 mL liquid LB medium supplemented with 100 mgL1 kanamycin and grown at 250 rpm, 30 °C to an OD of 0.6–1.0, and kept overnight at +4 °C. Subsequently, 4 mL obtained culture was used to inoculate a 400 mL pre-culture in a shake flask using the same medium and conditions as above. Cells were concentrated by centrifugation at 2000g for 15 min at +4 °C and resuspended in 25 mL fresh medium to inoculate the fermentor. When the dioxygen consumption started to decrease (after approximately 8 h) exponential feeding of feed media I and II was initiated simultaneously. Prior to depletion of glucose (after a total cultivation time of approximately 44 h), cells were harvested by centrifugation at 3,000g for 15 min at +4 °C and finally stored at )80 °C until isolation and purification of the protein was initiated. Determination of optical density and dry-cell weights Cell growth was followed by measurement of the optical density at 600 nm. Dry-cell weights (DCW) were determined from 1 mL aliquots of culture medium, as described by Korz et al. [22]. Purification of recombinant Pseudomonas stutzeri cytochrome c4 Approximately 250 g cell paste was thawed on ice and resuspended in ice-cold 0:5 mM MgCl2 . The red/orange suspension was diluted with Milli-Q water to a conductivity of below 2:5 mScm1 and the vast majority of cell debris was removed by adding a small aliquot of a DE52 anion-exchanger (Whatman) pre-equilibrated with 5 mM Tris–HCl pH 7.5. Batch adsorption of the protein was achieved by addition of more anion-exchanger resin. The red/orange recombinant cytochrome c4 was eluted with 5 mM Tris–HCl, 0.15 M NaCl, pH 7.5. Lowering of the salt concentration of the protein elute was accomplished by ultrafiltration (using a 200 mL stirred Amicon cell fitted with a YM3 membrane), followed by dilution with water. Next, the protein solution was lyophilized and stored at )20 °C for further purification. A minimum volume of 0:2 M NH4 HCO3 was used to dis€ KTA purifier solve the protein before gel filtration on a A (Amersham Biosciences) equipped with a Superdex 75 column (Amersham Biosciences) equilibrated with 0:2 M NH4 HCO3 . Fractions containing cytochrome c4 , as monitored at 416 nm, were pooled and lyophilized. Following dissolution in 5 mM Tris–HCl, pH 7.5, cytochrome c4 was purified on a Source 30Q column (Amersham Biosciences) using a linear gradient of 0–0.125 M NaCl in 5 mM Tris–HCl, pH 7.5. Fractions containing reduced, half-reduced, and oxidized cytochrome c4 were collected and oxidized by ½FeðCNÞ6 3 , and the excess oxidant was removed by ultrafiltration. The oxidized
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cytochrome c4 was purified further on the Source 30Q column using the same linear gradient as above. The eluted fractions of oxidized recombinant cytochrome c4 were pooled and reduced with sodium ascorbate. The excess reductant was removed by ultrafiltration, as described above. Subsequently, the reduced recombinant cytochrome c4 was purified on a Source 30Q column using the same linear gradient as above. Finally, the purified protein was exchanged into 0:2 M NH4 HCO3 on a HiPrep 26/10 desalting column (Amersham Biosciences), lyophilized, and stored at )20 °C until further use. Protein gel electrophoresis The progress of the recombinant cytochrome c4 purification was followed by SDS–PAGE using a pre-cast 15% Tris–glycine gel, a mini-PROTEAN II cell, and polypeptide markers (Bio-rad). Protein bands were stained with Coomassie blue G-250 dye. Electronic absorption spectroscopy The UV–vis spectra were recorded on a HP8453 (Agilent Technologies) diode-array spectrophotometer at room temperature. Mass spectrometry The mass spectra were acquired on a Nanoflow ESI Q-TOF MS instrument from Micromass, UK. It was operated according to manufacturerÕs recommendation with a needle and cone voltage of 850 and 25 V, respectively. An Au/Pd-coated borosilicate glass capillary tip (cat. No. ES380, MDS Proteomics, Denmark) was used. The mass spectrum was obtained from the m=zspectrum using the transform algorithm provided by the MassLynx v3.4 software (Micromass). Electrochemistry Cyclic voltammetry measurements were performed with an Eco Chemie Autolab potentiostat equipped with an ECD low-current module. The working electrode was a 4,40 -bipyridyl disulfide modified gold electrode prepared by dipping a freshly polished and sonicated gold electrode for approximately 6 min in a 0.5 mM promoter solution, followed by careful flushing with pure buffer. The twocompartment electrochemical cell and details of the electrochemical procedures have been described previously [23].
Results and discussion We have successfully expressed P. stutzeri cytochrome c4 in P. putida PaW340 cells transformed with
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the pNM185 plasmid containing the 0.7 kbp DNA sequence consisting of (a) a 39-bp sequence upstream from the ATG start codon containing the native ribosomebinding site, (b) the sequence encoding the native signal peptide sequence, and (c) the coding sequence of the mature cytochrome c4 . Expression of cytochrome c4 is activated by the m-toluate inducible xylS=Pm promoter on the pNM185 plasmid [17]. Mature cytochrome c4 cleaved from the signal peptide is obtained (see below). However, the yield of purified cytochrome c4 from cells grown in shake flask culture is moderate at 4–5 mg/L of culture and comparable to what has been obtained for most other c-type cytochromes [8–11]. We have therefore, developed an exponential feeding fed-batch cultivation procedure for the production of recombinant P. stutzeri cytochrome c4 in P. putida. After approximately 8 h of batch cultivation, the initial glucose is depleted and the exponential feeding of glucose is initiated. After approximately 44 h of cultivation (in total), cells are harvested. The DCW at the time of harvest is typically 34 g DCW/L and the yield of wet-cell paste is typically 500 g from the approximately 4 L culture medium. With the current experimental settings, the cell growth rate is not limited by the dissolved dioxygen concentration and the final yield of purified cytochrome c4 is approximately 29 mg/L of culture. The described strategy constitutes the first example where high-cell-density cultivation of P. putida is successfully used for production of a recombinant protein. In the native source, cytochrome c4 is located in the periplasm, partly associated with the periplasmic face of the cell membrane [24]. Consequently, when purifying cytochrome c4 from non-recombinant bacteria, the protein is completely extracted from the cells using an organic co-solvent (n-butanol) [24]. However, our data show that the increased level of cytochrome c4 produced in recombinant bacteria accumulates in a non-membrane associated form in the periplasmic space and is easy released using MgCl2 . This is supported by the fact that utilization of n-butanol did not increase the yield of recombinant cytochrome c4 (data not shown).
Fig. 1. Purification of recombinant P. stutzeri cytochrome c4 followed by SDS–PAGE. Lane 1: Polypeptide markers from Bio-Rad. 25.6, 19.7, 14.4, 6.5, and 3.5 kDa. Lane 2: Five times concentrated MgCl2 solution protein extract. Lane 3: Eluate from the DE52 anion-exchange column. Lane 4: Pooled fractions from the Superdex 75 gel filtration column. Lane 5: Pooled fractions from the first Source 30Q anion-exchange column. Lane 6: Oxidized, recombinant P. sturzeri cytochrome c4 fraction from the second Source 30Q anion-exchange column. Lane 7: Reduced, recombinant P. stutzeri cytochrome c4 fraction from the third Source 30Q anion-exchange column.
The purification strategy follows a traditional capture, purification, and polishing scheme. The progress of purification, as monitored by SDS–PAGE, is shown in Fig. 1 and the numerical results of the purification are summarized in Table 1. To remove traces of impurities that co-elute with cytochrome c4 , the possibility of concurrent change of oxidation state and change in elution pattern was exploited. Hence, ion-exchange chromatography of cytochrome c4 in the oxidized as well as in the reduced form was performed on the Source 30Q column. This method of polishing follows the procedure commonly used for non-recombinant cytochrome c4 . The electronic absorption spectrum of purified reduced recombinant P. stutzeri cytochrome c4 is shown in Fig. 2. The purified recombinant protein has a ‘‘protein purity ratio’’ A280 =A416 of 0.112 and a ‘‘cytochrome
Table 1 Summary of purification of recombinant P. stutzeri cytochrome c4 a Purification step
Proteinb (g)
Cytochrome c4 c (mg)
Yield (%)
Protein purity A280 =A416
Extract DE52 Superdex 75 1. Source 30Q 2. Source 30Q ox 3. Source 30Q re
13
270 209 170 124 118 115
100 77 63 46 44 43
7.38 1.12 0.373 0.239
a
—
0.112
Cell paste (500 g wet cells) from a 4 L high-cell-density fed-batch cultivation of P. putida. The amount of protein in the extract was calculated from the absorbance at 280 nm according to A1% 280 ¼ 10 [25]. c Calculated using a 550 nm extinction coefficient of 44; 400 M1 cm1 for reduced cytochrome c4 [24]. d For reduced cytochrome c4 . b
Cytochrome purityd A550 =A522
1.14
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Fig. 2. Electronic absorption spectroscopy of reduced, recombinant P. stutzeri cytochrome c4 (2:8 lM) in 5 mM Tris–HCl, 0.1 M NaCl, pH 7.5.
purity ratio’’ A550 =A522 of 1.14, which are essentially identical to the values for the native protein purified from P. stutzeri [23]. In addition, recombinant cytochrome c4 can be crystallized under the conditions previously reported for the native protein [26]. Data will be presented elsewhere. For the mass spectrometric discussion, we use the terms ‘‘apparent mass,’’ ‘‘calculated mass’’, and ‘‘molecular weight,’’ as defined in [27]. The calculated mass is the sum of the molecular weight of the protein part (19,679.40 Da as based on the amino acid sequence), the two protoporphyrin IX groups and the two iron-ions (2x(562.68 Da+ 55.847 Da)), giving in total a calculated mass for ‘‘neutral’’ cytochrome c4 of 20,916.5 Da. To obtain the theoretical apparent mass for the oxidized form of the protein, the molecular weight of two times three protons has to be subtracted, thus, giving a theoretical apparent mass of 20,910.4 Da. Fig. 3 shows the mass spectrum of recombinant cytochrome c4 with charge states from +13 to +10. From these data, an apparent mass of 20,911 Da is obtained, which is in excellent agreement with the theoretical apparent mass. Mass spectrometric analysis of cytochrome c4 unfolded by formic acid or digested with trypsin also confirmed the covalent attachment of the heme groups to the protein (data not shown). The functional properties of the recombinant cytochrome c4 were assessed by cyclic voltammetry at promoter modified gold electrodes. As shown in Fig. 4, the voltammetric signal shows two close lying peaks in both scan directions, which indicates that both heme groups are electrochemically active. The reduction potentials
Fig. 3. Nano-electrospray ionization time-of-flight mass spectrometry analysis of purified recombinant P. stutzeri cytochrome c4 . The sample was desalted prior to analysis and sprayed from water. The concentration was approximately 20 lM. The upper panel shows the positive ion-mode mass over charge (m=z) spectrum. The lower panel shows the transformed mass spectrum (Da). The theoretical apparent mass is 20,910.4 Da.
for the two heme groups are 230 and 332 mV vs. SHE, respectively. This is in agreement with the corresponding data for native cytochrome c4 , cf. our previous studies [23]. This work constitutes the first successful production of a recombinant c-type di-heme protein. The properties of the purified recombinant protein are indistinguishable from those of native P. stutzeri cytochrome c4 , as shown by a number of different characterization techniques.
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technical assistance of Lise–Lotte Jespersen and Louise Rosgaard is acknowledged. This work was supported by a Danish Natural Science Research Council grant to M.H.T., a Ph.D.-scholarship from the Technical University of Denmark to A.N., a Danish Technical Research Council grant and a Carlsberg Foundation grant to H.E.M.C, and a grant from the Brdr. Hartmann foundation.
References
Fig. 4. Cyclic voltammogram (upper panel) of oxidized recombinant P. stutzeri cytochrome c4 (200 lM) in 100 mM phosphate buffer, pH 7.5, on a 4,40 -bipyridyl disulfide modified gold electrode. Scan rate 10 mV/s. Lower panel shows the first derivative of the voltammogram.
Furthermore, the protein has been expressed in high yield by successful development of a strategy using highcell-density fed-batch cultivation of P. putida. This demonstrates that P. putida is an appropriate bacterial organism for effective production of recombinant prokaryotic metalloproteins. In conclusion, the described high-yield fermentation and rapid isolation procedure will greatly enhance and facilitate biochemical characterization of both wild-type and mutant derivatives of cytochrome c4 .
Acknowledgments We thank Professor Kenneth Timmis for the generous gift of the pNM185 expression plasmid. We greatly appreciate the assistance from Dr. Ole Nørregaard Jensen with the mass spectrometry analysis, which was performed at the Danish Mass Spectrometry Instrument Center, University of Southern Denmark. The skilled
[1] N.H. Andersen, H.E.M. Christensen, G. Iversen, A. Nørgaard, C. Scharnagl, M.H. Thuesen, J. Ulstrup, Cytochrome c4 , in: A. Messerschmidt, R. Huber, T. Poulos, K. Wieghardt (Eds.), Handbook of Metalloproteins, Wiley, Sussex, UK, 2001, pp. 100–109. [2] A. Kadziola, S. Larsen, Crystal structure of the dihaem cyto resochrome c4 from Pseudomonas stutzeri determined at 2.2 A lution, Structure 5 (1997) 203–216. [3] Z.W. Chen, M. Koh, G. Van Driessche, J.J. Van Beeumen, R.G. Bartsch, T.E. Meyer, M.A. Cusanovich, F.S. Mathews, The structure of flavocytochrome c sulfide dehydrogenase from a purple phototrophic bacterium, Science 266 (1994) 430–432. [4] B.A. Averill, Dissimilatory nitrite and nitric oxide reductases, Chem. Rev. 96 (1996) 2951–2964. [5] V. F€ ul€ op, C.J. Ridout, C. Greenwood, J. Hajdu, Crystal structure of the di-haem cytochrome c peroxidase from Pseudomonas aeruginosa, Structure 3 (1995) 1225–1233. [6] J.M. Moulis, L.C. Sieker, K.S. Wilson, Z. Dauter, Crystal structure of the 2[4Fe–4S] ferredoxin from Chromatium vinosum: Evolutionary and mechanistic inferences for [3/4Fe–4S] ferredoxins, Protein Sci. 5 (1996) 1765–1775. [7] G.R. Moore, G.W. Pettigrew, Cytochromes c: evolutionary, structural, and physiological aspects, Springer-Verlag, Berlin, 1990. [8] W.B. Pollock, F.I. Rosell, M.B. Twitchett, M.E. Dumont, A.G. Mauk, Bacterial expression of a mitochondrial cytochrome c. Trimethylation of lys72 in yeast iso-1-cytochrome c and the alkaline conformational transition, Biochemistry 37 (1998) 6124–6131. [9] E. Arslan, H. Schulz, R. Zufferey, P. K€ unzler, L. Th€ ony-Meyer, Overproduction of the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb3 oxidase in Escherichia coli, Biochem. Biophys. Res. Commun. 251 (1998) 744–747. [10] B. Reincke, L. Th€ ony-Meyer, C. Dannehl, A. Odenwald, M. Aidim, H. Witt, H. R€ uterjans, B. Ludwid, Heterologous expression of soluble fragments of cytochrome c552 acting as electron donor to the Paracoccus denitrificans cytochrome c oxidase, Biochem. Biophys. Acta 1411 (1999) 114–120. [11] P.D. Barker, I. Bertini, R. Del Conte, S.J. Ferguson, P. Hajieva, E. Tomlinson, P. Turano, M.S. Viezzoli, A further clue to understanding the mobility of mitochondrial yeast cytochrome c, Eur. J. Biochem. 268 (2001) 4468–4476. [12] F. Cutruzzola, I. Ciabatti, G. Rolli, S. Falcinelli, M. Arese, G. Rangohino, A. Anselmino, E. Zennaro, M.C. Silvestrini, Expression and characterization of Pseudomonas aeruginosa cytochrome c-551 and two site-directed mutants: role of tryptophan 56 in the modulation of redox properties, Biochem. J. 322 (1997) 35–42. [13] H.E.M. Christensen, Cloning and characterisation of the gene encoding cytochrome c4 from Pseudomonas stutzeri, Gene 144 (1994) 139–140. [14] S.Y. Lee, High-cell-density culture of Escherichia coli, Trends Biotechnol. 14 (1996) 98–105.
M.H. Thuesen et al. / Protein Expression and Purification 27 (2003) 175–181 [15] D. Riesenberg, R. Guthke, High-cell-density cultivation of microorganisms, Appl. Microbiol. Biotechnol. 51 (1999) 422– 430. [16] G.J. Kim, I.Y. Lee, D.K. Choi, S.C. Yoon, Y.H. Park, Highdensity cultivation of Pseudomonas putida BM01 using glucose, J. Microbiol. Biotechnol. 6 (1996) 221–224. [17] N. Mermod, J.L. Ramos, P.R. Lehrbach, K.N. Timmis, Vector for regulated expression of cloned genes in a wide range of Gramnegative bacteria, J. Bacteriol. 167 (1986) 447–454. [18] F.C. Franklin, P.A. Williams, Construction of a partial diploid for the degradative pathway encoded by the TOL plasmid (pWWO) from Pseudomonas putida mt-2: evidence for the positive nature of the regulation by the xyIR gene, Mol. Gen. Genet. 177 (1980) 321–328. [19] J.H. Cho, E.K. Kim, J.S. So, Improved transformation of Pseudomonas putida KT2440 by electroporation, Biotechnol. Tech. 9 (1995) 41–44. [20] N. Itoh, T. Kousai, Y. Koide, Efficient transformation of Pseudomonas strains with pNI vectors by electroporation, Biosci. Biotechnol. Biochem. 58 (1994) 1306–1308. [21] C.S. Shin, M.S. Hong, D.Y. Kim, J. Lee, Y.H. Park, Synthesis of mini-proinsulin precursors using N-termini of human TNF-a as
[22]
[23]
[24] [25]
[26]
[27]
181
fusion partners in recombinant Escherichia coli, J. Ind. Microbiol. Biotechnol. 22 (1999) 176–180. D.J. Korz, U. Rinas, K. Hellmuth, E.A. Sanders, W.D. Deckwer, Simple fed-batch techniques for high-cell-density cultivation of Escherichia coli, J. Biotechnol. 39 (1995) 59–65. J.J. Karlsson, M.F. Nielsen, M.H. Thuesen, J. Ulstrup, Electrochemistry of cytochrome c4 from Pseudomonas stutzeri, J. Phys. Chem. B 101 (1997) 2430–2436. G.W. Pettigrew, K.R. Brown, Free and membrane-bound forms of bacterial cytochrome c4 , Biochem. J. 252 (1988) 427–435. C.N. Cronin, W.S. McIntire, Heterologous expression in Pseudomonas aeruginosa and purification of the 9.2-kDa c-type cytochrome subunit of p-cresol methylhydroxylase, Protein Expr. Purif. 19 (2000) 74–83. A. Kadziola, S. Larsen, H.E.M. Christensen, J.J. Karlsson, J. Ulstrup, Crystallization and preliminary crystallographic investigations of cytochrome c4 form Pseudomonas stutzeri, Acta Crystallogr. D 51 (1995) 1071–1073. K.A. Johnson, M.F. Verhagen, P.S. Brereton, M.W. Adams, I.J. Amster, Probing the stoichiometry and oxidation states of metal centers in iron–sulfur proteins using electrospray FTICR mass spectrometry, Anal. Chem. 72 (2000) 1410–1418.
Expression of recombinant Pseudomonas stutzeri di-heme cytochrome c4 by high-cell-density fed-batch cultivation of Pseudomonas putida Marianne H. Thuesen, Allan Nørgaard, Anne M. Hansen, Mikael B. Caspersen, and Hans E.M. Christensen* Department of Chemistry, Building 207, Technical University of Denmark, 2800 Lyngby, Denmark Received 16 July 2002, and in revised form 20 August 2002
Abstract The gene of the di-heme protein cytochrome c4 from Pseudomonas stutzeri was expressed in Pseudomonas putida. High-yield expression of the protein was achieved by high-cell-density fed-batch cultivation using an exponential glucose feeding strategy. The recombinant cytochrome c4 protein was purified to apparent homogeneity and analyzed by electronic absorption spectroscopy, nanoflow electrospray ionization time-of-flight mass spectrometry, and electrochemistry. Cyclic voltammograms and UV–vis electronic absorption spectra were indistinguishable from the equivalent data of native P. stutzeri cytochrome c4 . Furthermore, the calculated and experimentally determined molecular masses of recombinant cytochrome c4 were identical. Biochemical characterization of both wild-type and mutant derivatives of the protein will be greatly enhanced and facilitated by the described high-yield fermentation and rapid isolation procedure. Ó 2002 Elsevier Science (USA). All rights reserved.
Cytochrome c4 is a 20 kDa bacterial di-heme electron transfer protein [1]. As revealed by X-ray crystal structures, the protein consists of two similarly folded domains, each containing a heme group [2,3]. Two-center metalloproteins are the simplest representative of multicenter metalloproteins and can thus serve as a model system for studying cooperative features in electron transfer proteins with a more complicated kinetic scheme [1]. In this context, studies of site-directed mutants are expected to give important information on the relationship between structure and cooperative behavior. Besides cytochrome c4 , only a few other twocenter metalloproteins are known, e.g., cytochrome cd1 nitrite reductase [4], cytochrome c peroxidase [5], the copper containing nitrite reductase [4], and certain ferredoxins [6]. Cytochrome c4 belongs to the class of c-type cytochromes [7] in which the heme groups are covalently *
Corresponding author. Fax: +45-45-88-31-36. E-mail address: [email protected] (H.E.M. Christensen). URL: http://www.kemi.dtu.dk.
attached to the protein via bonds to two cysteines. The covalent attachment of the heme group is believed to be the cause of the variable success of expression of c-type cytochromes in Escherichia coli as compared to other heme proteins [8]. However, the problem of expression of c-type cytochromes in E. coli has recently been overcome by coexpression of either yeast cytochrome c heme lyase [8] or of the eight cytochrome c maturation genes, the so-called ccm genes [9–11]. Another approach for expression of a c-type cytochrome (Pseudomonas aeruginosa cytochrome c-551) is based on the combination of the pNM185 expression plasmid and the Pseudomonas putida PaW340 strain as an expression host [12]. Several cytochrome c4 genes have been cloned and sequenced [1] including the gene encoding the Pseudomonas stutzeri cytochrome c4 [13], but an expression system for production of recombinant cytochrome c4 has so far not been reported. The subject of high-cell-density cultivation of microorganisms was reviewed recently [14,15]. The aim of the technique is to increase the production of the compound of interest per unit volume. In a study of the production
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of poly(3-hydroxyalkanoates), it was shown that P. putida grows efficiently to high-cell-density on glucose as a sole carbon source [16], but there has been no report on high-cell-density cultivation of P. putida for recombinant protein production. Here, we report on expression of recombinant P. stutzeri cytochrome c4 by high-cell-density fed-batch cultivation of P. putida, purification of the recombinant cytochrome c4; and its initial characterization by a number of chemical and physical techniques.
Materials and methods Synthetic oligonucleotides, LB media, Yeast Extract, and Peptone 140 were obtained from Life Technologies. Glucose (G5400) and d-aminolevulinic acid–HCl (A3785) were obtained from Sigma–Aldrich. An m-toluate stock solution with a nominal concentration of 100 mM was prepared from m-toluic acid (Sigma–Aldrich, T3,660-9) by first grinding the chemical in a mortar with a pestle and then suspending it in 1 M NaOH, followed by sonication for an extended period of time. Subsequently, the pH was adjusted to 7.5 with 1 M HCl. The resulting yellowish milky suspension was filtered through a 0:45 lm HPLC buffer filtration system and finally sterilized using a 0:2 lm sterile filter. All other chemicals were of analytical grade and solutions were prepared using 18:2 MX cm QPAK Milli-Q water (Millipore). Construction of cytochrome c4 expression vector The P. stutzeri cytochrome c4 gene has previously been cloned on a 3.5-kbp DNA fragment [13]. The original EcoRI restriction enzyme cleavage site was maintained and a BamHI and a SacI restriction enzyme site were introduced after nucleotide number 696 by the use of PCR. Using the protocol provided by the manufacturer, the 0.7kbp gene sequence was amplified with Pyrococcus furiosus DNA polymerase (Stratagene) and cloned into the pPCRScript Amp SK vector (Stratagene). The DNA sequence was confirmed using a BigDye terminator chemistry protocol (Applied Biosystems) and analyzed on an ABI310 (Applied Biosystems) DNA sequencing instrument. The cytochrome c4 encoding EcoRI/SacI DNA fragment was ligated with the EcoRI/SacI restriction enzyme digested pNM185 expression plasmid [17]. Pseudomonas Putida PaW340 cells [18] (DSM, strain No. 2112) were transformed with the resulting cytochrome c4 expression plasmid by electroporation essentially as described in [19,20]. The PaW340 strain was cultured in LB medium supplemented with 1000 mgL1 streptomycin at 30 °C. Inactivation of the streptomycin resistance gene on the pNM185 plasmid was accomplished by insertion of the cytochrome c4 gene. For selection of the pNM
185 plasmid, the LB media were supplemented with 100 mgL1 kanamycin. High-cell-density cultivation procedure A Biostat B fermentor with a 5 L autoclavable jacketed glass vessel (B. Braun Biotech International) equipped with a Mettler Toledo dissolved dioxygen sensor and a Mettler Toledo gel combination pH electrode was used. Prior to autoclaving, the pH electrode was calibrated. During cultivation, the temperature was maintained at 30 °C and the pH at 6:75 0:1 by PIDcontrolled addition of 6 M ammonium hydroxide. Adjustment of the zero-point and the 100% setting of the dioxygen electrode were performed after flushing the fermentor with dinitrogen and with a 5 L/min flow rate of air at a stirrer speed of 800, respectively. The level of dissolved dioxygen was maintained at 40% by using a double cascade in which the stirrer speed was first increased to a maximum of 800 rpm, followed by a gasmix-mode where the airflow was enriched with pure dioxygen to maintain the required level of dissolved dioxygen in the medium. The gas flow was maintained at 5 L/min. Antifoam 289 (Sigma–Aldrich, A5551) was added to a concentration of 20 ppm at the onset of the fermentation and then automatically added as required throughout the fermentation. The semi-synthetic 2.5 L initial batch medium contained (a) 32:5 g KH2 PO4 , 10 g ðNH4 Þ2 HPO4 , 5 g NH4 SO4 , 10 g yeast extract, 4.25 g citric acid, and 50 ml trace element stock solution [21]; (b) 3:0 g MgSO4 7H2 O; (c) 50 g D (+)-glucose; (d) 0.25 g thiamine–HCl; (e) 1 mM m-toluate; (f) 0.1 mM d-aminolevulinic acid; and (g) 100 mgL1 kanamycin. Components (a)–(c) were autoclaved separately, while components (d)–(g) were added as filter sterilized solutions. The recipe of the medium is partly based on the work of Shin et al. [21]. Feed medium I (1 L) contained (a) 800 g D (+)-glucose; (b) 2:0 g MgSO4 7H2 O; (c) 2:0 g ðNH4 Þ2 SO4 ; (d) 20 ml trace element stock solution [21]; (e) 1 mM mtoluate; (f) 0.1 mM d-aminolevulinic acid; and (g) 100 mgL1 kanamycin. Components (a)–(c) were autoclaved separately, while components (d)–(g) were added as filter sterilized solutions. Feed medium II (0.5 L) contained (a) 50 g yeast extract; (b) 100 g peptone 140; (c) 0:5 g MgSO4 7H2 O; (d) 1 mM m-toluate; (e) 0.1 mM d-aminolevulinic acid; and (f) 100 mgL1 kanamycin. Components (a)–(b) were autoclaved separately, while components (c)–(f) were added as filter sterilized solutions. Pre-cultures were grown overnight from )80 °C glycerol stocks on Luria–Bertini (LB) medium agar plates supplemented with 100 mgL1 kanamycin at 30 °C. A single colony was used to inoculate 3 mL liquid LB medium supplemented with 100 mgL1 kanamycin grown at 250 rpm, 30 °C to an OD of 0.6–1.0, and kept
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overnight at +4 °C. Then 0.5 mL was used to inoculate 50 mL liquid LB medium supplemented with 100 mgL1 kanamycin and grown at 250 rpm, 30 °C to an OD of 0.6–1.0, and kept overnight at +4 °C. Subsequently, 4 mL obtained culture was used to inoculate a 400 mL pre-culture in a shake flask using the same medium and conditions as above. Cells were concentrated by centrifugation at 2000g for 15 min at +4 °C and resuspended in 25 mL fresh medium to inoculate the fermentor. When the dioxygen consumption started to decrease (after approximately 8 h) exponential feeding of feed media I and II was initiated simultaneously. Prior to depletion of glucose (after a total cultivation time of approximately 44 h), cells were harvested by centrifugation at 3,000g for 15 min at +4 °C and finally stored at )80 °C until isolation and purification of the protein was initiated. Determination of optical density and dry-cell weights Cell growth was followed by measurement of the optical density at 600 nm. Dry-cell weights (DCW) were determined from 1 mL aliquots of culture medium, as described by Korz et al. [22]. Purification of recombinant Pseudomonas stutzeri cytochrome c4 Approximately 250 g cell paste was thawed on ice and resuspended in ice-cold 0:5 mM MgCl2 . The red/orange suspension was diluted with Milli-Q water to a conductivity of below 2:5 mScm1 and the vast majority of cell debris was removed by adding a small aliquot of a DE52 anion-exchanger (Whatman) pre-equilibrated with 5 mM Tris–HCl pH 7.5. Batch adsorption of the protein was achieved by addition of more anion-exchanger resin. The red/orange recombinant cytochrome c4 was eluted with 5 mM Tris–HCl, 0.15 M NaCl, pH 7.5. Lowering of the salt concentration of the protein elute was accomplished by ultrafiltration (using a 200 mL stirred Amicon cell fitted with a YM3 membrane), followed by dilution with water. Next, the protein solution was lyophilized and stored at )20 °C for further purification. A minimum volume of 0:2 M NH4 HCO3 was used to dis€ KTA purifier solve the protein before gel filtration on a A (Amersham Biosciences) equipped with a Superdex 75 column (Amersham Biosciences) equilibrated with 0:2 M NH4 HCO3 . Fractions containing cytochrome c4 , as monitored at 416 nm, were pooled and lyophilized. Following dissolution in 5 mM Tris–HCl, pH 7.5, cytochrome c4 was purified on a Source 30Q column (Amersham Biosciences) using a linear gradient of 0–0.125 M NaCl in 5 mM Tris–HCl, pH 7.5. Fractions containing reduced, half-reduced, and oxidized cytochrome c4 were collected and oxidized by ½FeðCNÞ6 3 , and the excess oxidant was removed by ultrafiltration. The oxidized
177
cytochrome c4 was purified further on the Source 30Q column using the same linear gradient as above. The eluted fractions of oxidized recombinant cytochrome c4 were pooled and reduced with sodium ascorbate. The excess reductant was removed by ultrafiltration, as described above. Subsequently, the reduced recombinant cytochrome c4 was purified on a Source 30Q column using the same linear gradient as above. Finally, the purified protein was exchanged into 0:2 M NH4 HCO3 on a HiPrep 26/10 desalting column (Amersham Biosciences), lyophilized, and stored at )20 °C until further use. Protein gel electrophoresis The progress of the recombinant cytochrome c4 purification was followed by SDS–PAGE using a pre-cast 15% Tris–glycine gel, a mini-PROTEAN II cell, and polypeptide markers (Bio-rad). Protein bands were stained with Coomassie blue G-250 dye. Electronic absorption spectroscopy The UV–vis spectra were recorded on a HP8453 (Agilent Technologies) diode-array spectrophotometer at room temperature. Mass spectrometry The mass spectra were acquired on a Nanoflow ESI Q-TOF MS instrument from Micromass, UK. It was operated according to manufacturerÕs recommendation with a needle and cone voltage of 850 and 25 V, respectively. An Au/Pd-coated borosilicate glass capillary tip (cat. No. ES380, MDS Proteomics, Denmark) was used. The mass spectrum was obtained from the m=zspectrum using the transform algorithm provided by the MassLynx v3.4 software (Micromass). Electrochemistry Cyclic voltammetry measurements were performed with an Eco Chemie Autolab potentiostat equipped with an ECD low-current module. The working electrode was a 4,40 -bipyridyl disulfide modified gold electrode prepared by dipping a freshly polished and sonicated gold electrode for approximately 6 min in a 0.5 mM promoter solution, followed by careful flushing with pure buffer. The twocompartment electrochemical cell and details of the electrochemical procedures have been described previously [23].
Results and discussion We have successfully expressed P. stutzeri cytochrome c4 in P. putida PaW340 cells transformed with
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the pNM185 plasmid containing the 0.7 kbp DNA sequence consisting of (a) a 39-bp sequence upstream from the ATG start codon containing the native ribosomebinding site, (b) the sequence encoding the native signal peptide sequence, and (c) the coding sequence of the mature cytochrome c4 . Expression of cytochrome c4 is activated by the m-toluate inducible xylS=Pm promoter on the pNM185 plasmid [17]. Mature cytochrome c4 cleaved from the signal peptide is obtained (see below). However, the yield of purified cytochrome c4 from cells grown in shake flask culture is moderate at 4–5 mg/L of culture and comparable to what has been obtained for most other c-type cytochromes [8–11]. We have therefore, developed an exponential feeding fed-batch cultivation procedure for the production of recombinant P. stutzeri cytochrome c4 in P. putida. After approximately 8 h of batch cultivation, the initial glucose is depleted and the exponential feeding of glucose is initiated. After approximately 44 h of cultivation (in total), cells are harvested. The DCW at the time of harvest is typically 34 g DCW/L and the yield of wet-cell paste is typically 500 g from the approximately 4 L culture medium. With the current experimental settings, the cell growth rate is not limited by the dissolved dioxygen concentration and the final yield of purified cytochrome c4 is approximately 29 mg/L of culture. The described strategy constitutes the first example where high-cell-density cultivation of P. putida is successfully used for production of a recombinant protein. In the native source, cytochrome c4 is located in the periplasm, partly associated with the periplasmic face of the cell membrane [24]. Consequently, when purifying cytochrome c4 from non-recombinant bacteria, the protein is completely extracted from the cells using an organic co-solvent (n-butanol) [24]. However, our data show that the increased level of cytochrome c4 produced in recombinant bacteria accumulates in a non-membrane associated form in the periplasmic space and is easy released using MgCl2 . This is supported by the fact that utilization of n-butanol did not increase the yield of recombinant cytochrome c4 (data not shown).
Fig. 1. Purification of recombinant P. stutzeri cytochrome c4 followed by SDS–PAGE. Lane 1: Polypeptide markers from Bio-Rad. 25.6, 19.7, 14.4, 6.5, and 3.5 kDa. Lane 2: Five times concentrated MgCl2 solution protein extract. Lane 3: Eluate from the DE52 anion-exchange column. Lane 4: Pooled fractions from the Superdex 75 gel filtration column. Lane 5: Pooled fractions from the first Source 30Q anion-exchange column. Lane 6: Oxidized, recombinant P. sturzeri cytochrome c4 fraction from the second Source 30Q anion-exchange column. Lane 7: Reduced, recombinant P. stutzeri cytochrome c4 fraction from the third Source 30Q anion-exchange column.
The purification strategy follows a traditional capture, purification, and polishing scheme. The progress of purification, as monitored by SDS–PAGE, is shown in Fig. 1 and the numerical results of the purification are summarized in Table 1. To remove traces of impurities that co-elute with cytochrome c4 , the possibility of concurrent change of oxidation state and change in elution pattern was exploited. Hence, ion-exchange chromatography of cytochrome c4 in the oxidized as well as in the reduced form was performed on the Source 30Q column. This method of polishing follows the procedure commonly used for non-recombinant cytochrome c4 . The electronic absorption spectrum of purified reduced recombinant P. stutzeri cytochrome c4 is shown in Fig. 2. The purified recombinant protein has a ‘‘protein purity ratio’’ A280 =A416 of 0.112 and a ‘‘cytochrome
Table 1 Summary of purification of recombinant P. stutzeri cytochrome c4 a Purification step
Proteinb (g)
Cytochrome c4 c (mg)
Yield (%)
Protein purity A280 =A416
Extract DE52 Superdex 75 1. Source 30Q 2. Source 30Q ox 3. Source 30Q re
13
270 209 170 124 118 115
100 77 63 46 44 43
7.38 1.12 0.373 0.239
a
—
0.112
Cell paste (500 g wet cells) from a 4 L high-cell-density fed-batch cultivation of P. putida. The amount of protein in the extract was calculated from the absorbance at 280 nm according to A1% 280 ¼ 10 [25]. c Calculated using a 550 nm extinction coefficient of 44; 400 M1 cm1 for reduced cytochrome c4 [24]. d For reduced cytochrome c4 . b
Cytochrome purityd A550 =A522
1.14
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Fig. 2. Electronic absorption spectroscopy of reduced, recombinant P. stutzeri cytochrome c4 (2:8 lM) in 5 mM Tris–HCl, 0.1 M NaCl, pH 7.5.
purity ratio’’ A550 =A522 of 1.14, which are essentially identical to the values for the native protein purified from P. stutzeri [23]. In addition, recombinant cytochrome c4 can be crystallized under the conditions previously reported for the native protein [26]. Data will be presented elsewhere. For the mass spectrometric discussion, we use the terms ‘‘apparent mass,’’ ‘‘calculated mass’’, and ‘‘molecular weight,’’ as defined in [27]. The calculated mass is the sum of the molecular weight of the protein part (19,679.40 Da as based on the amino acid sequence), the two protoporphyrin IX groups and the two iron-ions (2x(562.68 Da+ 55.847 Da)), giving in total a calculated mass for ‘‘neutral’’ cytochrome c4 of 20,916.5 Da. To obtain the theoretical apparent mass for the oxidized form of the protein, the molecular weight of two times three protons has to be subtracted, thus, giving a theoretical apparent mass of 20,910.4 Da. Fig. 3 shows the mass spectrum of recombinant cytochrome c4 with charge states from +13 to +10. From these data, an apparent mass of 20,911 Da is obtained, which is in excellent agreement with the theoretical apparent mass. Mass spectrometric analysis of cytochrome c4 unfolded by formic acid or digested with trypsin also confirmed the covalent attachment of the heme groups to the protein (data not shown). The functional properties of the recombinant cytochrome c4 were assessed by cyclic voltammetry at promoter modified gold electrodes. As shown in Fig. 4, the voltammetric signal shows two close lying peaks in both scan directions, which indicates that both heme groups are electrochemically active. The reduction potentials
Fig. 3. Nano-electrospray ionization time-of-flight mass spectrometry analysis of purified recombinant P. stutzeri cytochrome c4 . The sample was desalted prior to analysis and sprayed from water. The concentration was approximately 20 lM. The upper panel shows the positive ion-mode mass over charge (m=z) spectrum. The lower panel shows the transformed mass spectrum (Da). The theoretical apparent mass is 20,910.4 Da.
for the two heme groups are 230 and 332 mV vs. SHE, respectively. This is in agreement with the corresponding data for native cytochrome c4 , cf. our previous studies [23]. This work constitutes the first successful production of a recombinant c-type di-heme protein. The properties of the purified recombinant protein are indistinguishable from those of native P. stutzeri cytochrome c4 , as shown by a number of different characterization techniques.
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technical assistance of Lise–Lotte Jespersen and Louise Rosgaard is acknowledged. This work was supported by a Danish Natural Science Research Council grant to M.H.T., a Ph.D.-scholarship from the Technical University of Denmark to A.N., a Danish Technical Research Council grant and a Carlsberg Foundation grant to H.E.M.C, and a grant from the Brdr. Hartmann foundation.
References
Fig. 4. Cyclic voltammogram (upper panel) of oxidized recombinant P. stutzeri cytochrome c4 (200 lM) in 100 mM phosphate buffer, pH 7.5, on a 4,40 -bipyridyl disulfide modified gold electrode. Scan rate 10 mV/s. Lower panel shows the first derivative of the voltammogram.
Furthermore, the protein has been expressed in high yield by successful development of a strategy using highcell-density fed-batch cultivation of P. putida. This demonstrates that P. putida is an appropriate bacterial organism for effective production of recombinant prokaryotic metalloproteins. In conclusion, the described high-yield fermentation and rapid isolation procedure will greatly enhance and facilitate biochemical characterization of both wild-type and mutant derivatives of cytochrome c4 .
Acknowledgments We thank Professor Kenneth Timmis for the generous gift of the pNM185 expression plasmid. We greatly appreciate the assistance from Dr. Ole Nørregaard Jensen with the mass spectrometry analysis, which was performed at the Danish Mass Spectrometry Instrument Center, University of Southern Denmark. The skilled
[1] N.H. Andersen, H.E.M. Christensen, G. Iversen, A. Nørgaard, C. Scharnagl, M.H. Thuesen, J. Ulstrup, Cytochrome c4 , in: A. Messerschmidt, R. Huber, T. Poulos, K. Wieghardt (Eds.), Handbook of Metalloproteins, Wiley, Sussex, UK, 2001, pp. 100–109. [2] A. Kadziola, S. Larsen, Crystal structure of the dihaem cyto resochrome c4 from Pseudomonas stutzeri determined at 2.2 A lution, Structure 5 (1997) 203–216. [3] Z.W. Chen, M. Koh, G. Van Driessche, J.J. Van Beeumen, R.G. Bartsch, T.E. Meyer, M.A. Cusanovich, F.S. Mathews, The structure of flavocytochrome c sulfide dehydrogenase from a purple phototrophic bacterium, Science 266 (1994) 430–432. [4] B.A. Averill, Dissimilatory nitrite and nitric oxide reductases, Chem. Rev. 96 (1996) 2951–2964. [5] V. F€ ul€ op, C.J. Ridout, C. Greenwood, J. Hajdu, Crystal structure of the di-haem cytochrome c peroxidase from Pseudomonas aeruginosa, Structure 3 (1995) 1225–1233. [6] J.M. Moulis, L.C. Sieker, K.S. Wilson, Z. Dauter, Crystal structure of the 2[4Fe–4S] ferredoxin from Chromatium vinosum: Evolutionary and mechanistic inferences for [3/4Fe–4S] ferredoxins, Protein Sci. 5 (1996) 1765–1775. [7] G.R. Moore, G.W. Pettigrew, Cytochromes c: evolutionary, structural, and physiological aspects, Springer-Verlag, Berlin, 1990. [8] W.B. Pollock, F.I. Rosell, M.B. Twitchett, M.E. Dumont, A.G. Mauk, Bacterial expression of a mitochondrial cytochrome c. Trimethylation of lys72 in yeast iso-1-cytochrome c and the alkaline conformational transition, Biochemistry 37 (1998) 6124–6131. [9] E. Arslan, H. Schulz, R. Zufferey, P. K€ unzler, L. Th€ ony-Meyer, Overproduction of the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb3 oxidase in Escherichia coli, Biochem. Biophys. Res. Commun. 251 (1998) 744–747. [10] B. Reincke, L. Th€ ony-Meyer, C. Dannehl, A. Odenwald, M. Aidim, H. Witt, H. R€ uterjans, B. Ludwid, Heterologous expression of soluble fragments of cytochrome c552 acting as electron donor to the Paracoccus denitrificans cytochrome c oxidase, Biochem. Biophys. Acta 1411 (1999) 114–120. [11] P.D. Barker, I. Bertini, R. Del Conte, S.J. Ferguson, P. Hajieva, E. Tomlinson, P. Turano, M.S. Viezzoli, A further clue to understanding the mobility of mitochondrial yeast cytochrome c, Eur. J. Biochem. 268 (2001) 4468–4476. [12] F. Cutruzzola, I. Ciabatti, G. Rolli, S. Falcinelli, M. Arese, G. Rangohino, A. Anselmino, E. Zennaro, M.C. Silvestrini, Expression and characterization of Pseudomonas aeruginosa cytochrome c-551 and two site-directed mutants: role of tryptophan 56 in the modulation of redox properties, Biochem. J. 322 (1997) 35–42. [13] H.E.M. Christensen, Cloning and characterisation of the gene encoding cytochrome c4 from Pseudomonas stutzeri, Gene 144 (1994) 139–140. [14] S.Y. Lee, High-cell-density culture of Escherichia coli, Trends Biotechnol. 14 (1996) 98–105.
M.H. Thuesen et al. / Protein Expression and Purification 27 (2003) 175–181 [15] D. Riesenberg, R. Guthke, High-cell-density cultivation of microorganisms, Appl. Microbiol. Biotechnol. 51 (1999) 422– 430. [16] G.J. Kim, I.Y. Lee, D.K. Choi, S.C. Yoon, Y.H. Park, Highdensity cultivation of Pseudomonas putida BM01 using glucose, J. Microbiol. Biotechnol. 6 (1996) 221–224. [17] N. Mermod, J.L. Ramos, P.R. Lehrbach, K.N. Timmis, Vector for regulated expression of cloned genes in a wide range of Gramnegative bacteria, J. Bacteriol. 167 (1986) 447–454. [18] F.C. Franklin, P.A. Williams, Construction of a partial diploid for the degradative pathway encoded by the TOL plasmid (pWWO) from Pseudomonas putida mt-2: evidence for the positive nature of the regulation by the xyIR gene, Mol. Gen. Genet. 177 (1980) 321–328. [19] J.H. Cho, E.K. Kim, J.S. So, Improved transformation of Pseudomonas putida KT2440 by electroporation, Biotechnol. Tech. 9 (1995) 41–44. [20] N. Itoh, T. Kousai, Y. Koide, Efficient transformation of Pseudomonas strains with pNI vectors by electroporation, Biosci. Biotechnol. Biochem. 58 (1994) 1306–1308. [21] C.S. Shin, M.S. Hong, D.Y. Kim, J. Lee, Y.H. Park, Synthesis of mini-proinsulin precursors using N-termini of human TNF-a as
[22]
[23]
[24] [25]
[26]
[27]
181
fusion partners in recombinant Escherichia coli, J. Ind. Microbiol. Biotechnol. 22 (1999) 176–180. D.J. Korz, U. Rinas, K. Hellmuth, E.A. Sanders, W.D. Deckwer, Simple fed-batch techniques for high-cell-density cultivation of Escherichia coli, J. Biotechnol. 39 (1995) 59–65. J.J. Karlsson, M.F. Nielsen, M.H. Thuesen, J. Ulstrup, Electrochemistry of cytochrome c4 from Pseudomonas stutzeri, J. Phys. Chem. B 101 (1997) 2430–2436. G.W. Pettigrew, K.R. Brown, Free and membrane-bound forms of bacterial cytochrome c4 , Biochem. J. 252 (1988) 427–435. C.N. Cronin, W.S. McIntire, Heterologous expression in Pseudomonas aeruginosa and purification of the 9.2-kDa c-type cytochrome subunit of p-cresol methylhydroxylase, Protein Expr. Purif. 19 (2000) 74–83. A. Kadziola, S. Larsen, H.E.M. Christensen, J.J. Karlsson, J. Ulstrup, Crystallization and preliminary crystallographic investigations of cytochrome c4 form Pseudomonas stutzeri, Acta Crystallogr. D 51 (1995) 1071–1073. K.A. Johnson, M.F. Verhagen, P.S. Brereton, M.W. Adams, I.J. Amster, Probing the stoichiometry and oxidation states of metal centers in iron–sulfur proteins using electrospray FTICR mass spectrometry, Anal. Chem. 72 (2000) 1410–1418.