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High-Level Expression inEscherichia coliof the Soluble, Catalytic Domain of Rat Hepatic Cytochromeb5Reductase
PROTEIN EXPRESSION AND PURIFICATION ARTICLE NO.
8, 41–47 (1996)
0072
High-Level Expression in Escherichia coli of the Soluble, Catalytic Domain of Rat Hepatic Cytochrome b5 Reductase1 Michael J. Barber* and Gregory B. Quinn Department of Biochemistry and Molecular Biology, University of South Florida, College of Medicine, and *Institute for Biomolecular Science and H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612
Received November 30, 1995, and in revised form March 5, 1996
A T7 expression system has been produced for the high-level production of the soluble, catalytic domain of rat hepatic cytochrome b5 reductase in Escherichia coli. The recombinant protein was purified to homogeneity using affinity chromatography on 5*-ADP agarose and gel exclusion chromatography and exhibited a molecular mass of approximately 30 kDa by polyacrylamide gel electrophoresis and a molecular mass of 30,588 by mass spectrometry. Direct sequencing of the initial 12 residues of the amino-terminus of the purified domain yielded the sequence MITLENPDIKYP, identical to that predicted from the DNA sequence. The domain incorporated a full complement of FAD with a visible absorption spectrum typical of a flavoprotein exhibiting maxima at 389 and 461 nm and a distinct shoulder at 485 nm. Addition of NADH to the protein resulted in an extensive bleaching of the visible spectrum. The recombinant domain retained both NADH:ferricyanide and NADH:cytochrome b5 reductase activities with Vmax of 48 and 26 mmol NADH consumed/min/nmol FAD, respectively, and Km of 6, 7, and 11 mM for NADH, ferricyanide, and cytochrome b5. Comparison of the activities obtained using NADH and NADPH indicated a substantial preference for NADH as the reducing substrate. The results indicate that the recombinant protein retains the physical and catalytic properties of the native protein and represents an excellent system for probing the role of specific amino acid residues using site-directed mutagenesis. q 1996 Academic Press, Inc.
Hepatic cytochrome b5 reductase (EC 1.6.2.2) catalyzes the rapid transfer of two reducing equivalents 1 This work was supported by grant GM 32696 from the National Institutes of Health.
from the physiological electron donor NADH to two molecules of cytochrome b5 (1,2). The production of reduced cytochrome b5 represents a vital step in a variety of redox reactions that occur on the cytoplasmic surface of the endoplasmic reticulum. Thus, cytochrome b5 reductase participate in a number of important processes including the elongation and desaturation of fatty acids (3,4), cholesterol biosynthesis (5), and xenobiotic metabolism (6). Cytochrome b5 reductase has been isolated from a variety of sources including human (7), bovine (8), porcine (9), and rat (10) liver. In all cases, the monomeric amphipathic protein (molecular mass, approximately 35 kDa) comprised two domains, a small (molecular mass, 3 kDa) hydrophobic N-terminal membrane-anchoring domain which serves to orient the catalytic site at the membrane–aqueous interface and to facilitate rapid intermolecular electron transfer and a large (molecular mass, 32 kDa) hydrophilic catalytic domain which contains a single FAD prosthetic group. Separation of the two domains by limited proteolysis has facilitated isolation of a truncated, soluble form of the reductase which retains NADH:cytochrome b5 reductase activity (11). In addition to the physiological NADH:cytochrome b5 reductase activity, the enzyme can also effectively utilize ferricyanide as an artificial electron acceptor. Kinetic characterizations using either cytochrome b5 or ferricyanide have demonstrated that the rate-limiting step with either acceptor involves hydride transfer from NADH to the enzyme-bound FAD followed by more rapid electron transfer to the oxidizing substrate (12,13). A heterologous expression system has been developed for the production of a recombinant form of the bovine enzyme (14) which was produced in the form of a fusion protein that contained the complete amino acid 41
1046-5928/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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BARBER AND QUINN
sequence of the native protein, comprising both the membrane-anchoring and catalytic flavin-containing domains. Isolation of this protein followed by mild tryptic digestion was used to release the flavopeptide which has facilitated examination of the role of specific amino acid residues using site-directed mutagenesis. Utilizing PCR2 and the full-length cDNA for the rat cytochrome b5 reductase, we have constructed a heterologous expression system in Escherichia coli, using the T7 promoter, that specifically results in the efficient production of the flavin-containing hydrophilic domain and which retains the catalytic properties of the native microsomal protein. MATERIALS AND METHODS
Materials. NADH, NADPH, NAD/, 5*-ADP–agarose, cytochrome c (horse heart, type VI), myoglobin (sperm whale), bovine serum albumin, and FAD were obtained from Sigma Chemical Co. (St. Louis, MO). MOPS (‘‘ultrol’’ grade) was obtained from Calbiochem (San Diego, CA). Sinapinic acid (3,5-dimethoxy-4-hydroxy cinnamic acid) was purchased from Aldrich Chemical Co. (Milwaukee, WI) and IPTG was obtained from RPI (Mt. Prospect, IL). Restriction enzymes (NdeI and SacI) were obtained from Promega (Madison, WI). The pT7Blue and pET23B vectors were obtained from Novagen (Madison, WI). Recombinant soluble, rat cytochrome b5 was produced as described by Beck-von Bodman et al. (15). Geneclean, LB, and agar media were obtained from Bio101 (Vista, CA). Nucleotide sequencing was performed at the Alzheimer’s Research Laboratory, H. Lee Moffitt Cancer Center and Research Institute. Construction of the expression plasmid pCTYB5R. A schematic diagram showing the steps involved in the construction of the expression plasmid pCYTB5R is shown in Fig. 1. Oligonucleotide primers were synthesized to specifically amplify the 810-bp region of the construct pGBK3 corresponding to the coding sequence for the C-terminal 269 residues of the rat cytochrome b5 reductase sequence that represented the soluble catalytic domain: primer 1 (amino-terminus)—(5*-3*) CAT ATG ATC ACC CTC GAG AAC CCC GAC; primer 2 (carboxyl-terminus)—(5*-3*) GAA TTC TCA GAA GGT GAA GCA TCG CTC C. Primer 1 also contained a unique NdeI restriction site (CAT ATC) to subsequently facilitate direct cloning of the PCR fragment 2 Abbreviations used: NADH:FR, NADH:ferricyanide reductase; NADH:BR, NADH:cytochrome b5 reductase; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; IPTG, isopropyl b-D-thioglucopyranoside; HPLC, high-performance liquid chromatography; FPLC, fast protein liquid chromatography; MALDI–TOF, matrix-assisted laser desorption ionization–time of flight; m, ionic strength; MOPS, 3-(N-morpholino)propanesulfonic acid.
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FIG. 1. Construction of the pCYTB5R expression plasmid. A schematic diagram is shown to illustrate the various steps used to construct the expression vector used for the production of recombinant rat cytochrome b5 reductase.
into the pET23b expression vector while primer 2 contained an EcoRI site to permit rapid replacement of the 3* region of the b5 reductase DNA sequence for future manipulations. The pET23b vector was selected since this is one of the smallest vectors available that contains a F1 origin enabling the production of the single-stranded template required for future site-directed mutagenesis studies. In addition, while the pET23b vector potentially encodes a poly(His) affinity tag at the carboxyl-end of the expressed protein, the PCR product included a natural stop codon for the cytochrome b5 reductase sequence that prevented the incorporation of the poly(His) tag. Amplifications were performed using 5 units of Taq polymerase in the presence of 11 Taq buffer containing 80 ng of template pGBK3 vector DNA, 1 mM concentrations of each synthetic primer, 5% DMSO, and 2.5 mM dNTPs. Amplification conditions involved an initial incubation at 957C for 5 min, followed by 30 cycles at 947C for 1 min and 727C for 2 min, and ending with a 7-min extension at 727C. The amplified product was isolated using agarose gel electrophoresis and Geneclean purification. The purified PCR fragment was cloned into the pT7Blue vector by virtue of the artifactual 3* dA nucleotide overhangs which Taq polymerase generates and which also generates a SacI site at the 3* end, to create the construct
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pCRB5R. Subsequently the cytochrome b5 reductase sequence was excised from this construct by a NdeI–SacI restriction digest and ligated into NdeI–SacI-digested pET23b vector to create the construct pCYTB5R. The fidelity of the amplified cytochrome b5 reductase DNA sequence was verified by machine sequencing (ALF sequencer, Pharmacia-LKB Biotechnology) the entire PCR product in the expression construct using fluoricene-tagged T7 promoter and T7 terminator primers. Cell culture and protein purification. E. coli cells harboring the expression construct pCYTB5R were grown at 377C to an OD600 of 0.6, following which recombinant protein expression was induced by addition of IPTG (0.4 mM) and the cells cultured for a further 5 h. The cells were pelleted by centrifugation (400g, 20 min), washed with lysis buffer, and disrupted by sonication. Ammonium sulfate was added to 50% saturation and the solution clarified by centrifugation (10,000g, 15 min). Additional ammonium sulfate was added to 90% saturation and the solution centrifuged (14,000g, 20 min). The resulting pellet was resuspended in a minimal volume of 10 mM phosphate buffer, containing 0.1 mM EDTA, pH 7.0, dialyzed against the same buffer and applied to a column of 5*ADP agarose (2.5 1 10 cm). The column was extensively washed to remove extraneous proteins and the cytochrome b5 reductase eluted with NADH (100 mM). Fractions exhibiting NADH:FR activity were pooled, concentrated by pressure filtration, and further purified using gel exclusion FPLC on Superdex 75 (1 1 30 cm). Fractions containing cytochrome b5 reductance were pooled, concentrated, beaded, and stored under liquid N2 until required. Enzyme concentrations were estimated using A461 Å 10.6 cm01 mM01 and polyacrylamide gel electrophoresis was performed as described by Laemmli (16). Protein sequencing. Amino acid sequencing was performed as described by Neame and Barber (17) using an Applied Biosystems Intl. (Foster City, CA) 473A peptide sequencer. Mass spectrometry. Mass spectrometry was performed using a Kratos MALDI-III (Shimadzu Scientific Inst. Inc., Columbia, MD) mass spectrometer operating in the linear mode. Protein samples (10 pmol) in 0.1% trifluoroacetic acid were applied to sample slides in combination with the matrix, sinapinic acid. The spectrometer was calibrated using a mixture of cytochrome c (molecular mass, 12,384), myoglobin (molecular mass, 17,188), and bovine serum albumin (molecular mass, 66,524). Samples of purified FAD (molecular mass, 785) and FMN (molecular mass, 456) were used as prosthetic group standards. Enzyme activities. NADH:FR, NADPH:FR, and NADH:BR activities were determined at 257C under
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conditions of constant ionic strength and pH as previously described (18) in 116 mM MOPS buffer (m Å 0.05), containing 0.1 mM EDTA, pH 7.0. NADH:FR activity was determined as the decrease in absorbance at 340 nm in the presence of NADH (100 mM) and ferricyanide (200 mM). NADH:BR activity was determined as the increase in absorbance at 423 nm in the presence of NADH (100 mM) and soluble, recombinant rat cytochrome b5 (50 mM). All enzyme activities are expressed as initial rates for the oxidation of NADH (mmol of NADH consumed/ min/nmol FAD) or NADPH (mmol of NADPH consumed/min/nmol FAD), respectively. Initial rate data were analyzed using the software ‘‘ENZFIT’’ (Elsevier Biosoft, Ferguson, MO) to yield apparent Vmax and Km values. Spectroscopy. UV/visible spectra were obtained using a Shimadzu Scientific Inst. Inc. UV2101PC spectrophotometer. RESULTS
The results of a typical isolation of recombinant soluble, cytochrome b5 reductase are given in Table 1. Approximately 8 mg of enzyme was obtained starting from 1 liter of E. coli culture, corresponding to a yield of 43%. The purification procedure involved a minimal number of steps including ammonium sulfate fractionation, affinity chromatography on 5*-ADP–agarose, and FPLC size exclusion using Superdex 75. A typical elution profile obtained from the size exclusion chromatography is shown in Fig. 2 indicating the isolation of a single protein peak with NADH:FR activity. Polyacrylamide gel electrophoresis was used to examine the peak fraction obtained from the gel exclusion chromatography. A single protein band was detected, as shown in Fig. 3, that exhibited an approximate molecular weight of 30 kDa, in good agreement with the value predicted from the amino acid sequence of 30,576 Da. Mass spectrometry was utilized to accurately determine the molecular mass of the expressed soluble protein and to confirm the identity of the bound flavin prosthetic group. The mass spectrum obtained using MALDI–TOF is shown in Fig. 4. A peak corresponding to a mass/charge ratio of 30,588 was observed in the high-mass range corresponding to the M / H parent ion of the apoprotein together with a peak of substantially lower intensity at a mass/charge ratio of 15,321 corresponding to the M / 2H apoprotein ion. A peak of lower intensity was also apparent in the high-molecular-weight range that corresponded to a molecular mass of 31,391 which corresponds to the molecular mass of the M / H holoenzyme containing FAD. Shown in the inset is the mass spectrum obtained in the lowmass range with a peak corresponding to mass/charge
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BARBER AND QUINN TABLE 1
Purification of Recombinant Soluble, Rat Cytochrome b5 Reductase
Purification step
Activity (units)a
Protein (mg)
Specific activity (units/mg)
Recovery (%)
Fold
Cell supernatant 50% (NH4)2SO4 90% (NH4)2SO4 5*-ADP–agarose Superdex 75
26,247 19,340 18,627 12,762 11,211
320 93 72 10 8
82 208 259 1276 1400
100 74 71 49 43
1 3 3 16 17
a
Units: mmol NADH consumed/min.
ratios of 792 (M / H) corresponding to the molecular mass of FAD. The recombinant cytochrome b5 reductase was further analyzed using reverse-phase HPLC, as illustrated by the chromatogram shown in Fig. 5, prior to peptide sequencing. A single peak, eluting at 33.4 min, was obtained. Direct sequencing of 2 nmol of the protein yielded a methionine amino-terminal (1.5 nmol) followed by the residues Ile (1.4 nmol), Thr (0.3 nmol), Leu (1.3 nmol), Glu (0.8 nmol), Asn (1.2 nmol), Pro (1.0 nmol), Asp (1.1 nmol), Ile (0.8 nmol), Lys (1.3 nmol), Tyr (0.7 nmol), and Pro (0.9 nmol) for the first 12 cycles of direct sequencing. The additional small peak eluting at 5.2 min was subsequently identified as FAD by comparison with purified FAD and FMN standards. The UV/visible spectra obtained for the oxidized and reduced forms of the recombinant cytochrome b5 reductase are shown in Fig. 6. The spectrum of the oxidized domain was typical of a flavoprotein exhib-
iting absorption maxima in the visible range at 386 and 461 nm, together with a pronounced shoulder at 485 nm. Addition of NADH, under anaerobic conditions, resulted in a substantial bleaching of the visible spectrum and a corresponding decrease in the intensity in the UV region. Initial rate kinetic parameters obtained for the purified recombinant cytochrome b5 reductase catalytic domain are given in Table 2. Preliminary analysis of the domain’s catalytic activity indicated that neither the NADH:FR nor the NADH:BR activities were stimulated by the addition of exogenous FAD, suggesting that the domain contained a stoichiometric level of flavin chromophore. The results of a more extensive kinetic analysis revealed that all Lineweaver – Burke plots were linear over the various ranges of NADH, NADPH, ferricyanide, or cytochrome b5 concentrations examined. Under conditions of constant pH (7.0) and ionic strength (m Å 0.05), maximal NADH:FR ac-
FIG. 2. FPLC size exclusion chromatography of soluble, rat cytochrome b5 reductase. FPLC gel filtration was performed using a Superdex 75 column (1 1 30 cm) in 10 mM phosphate buffer, containing 0.1 mM EDTA, pH 7.0. Absorbance at 461 nm (.); absorbance at 280 nm (l); NADH:FR activity (j, mmol NADH consumed/min/ ml).
FIG. 3. Polyacrylamide gel electrophoresis of soluble, rat cytochrome b5 reductase. Recombinant cytochrome b5 reductase (3 mg protein, lane B) obtained following Superdex 75 gel filtration chromatography was analyzed on a 15% polyacrylamide gel. Lanes A and C correspond to protein molecular weight standards.
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FIG. 4. Mass spectrometry of recombinant cytochrome b5 reductase. The MALDI–TOF mass spectrum of purified recombinant soluble, rat cytochrome b5 reductase (10 pmol) was obtained in the presence of the matrix, sinapinic acid. The spectrum corresponds to the average obtained from 100 individual laser shots. The inset shows the mass spectrum of the low-mass region to indicate the presence of FAD.
tivity corresponded to 48 mmol NADH consumed/min/ nmol FAD while the corresponding value obtained for the NADH:BR activity was 24 mmol NADH consumed/ min/nmol FAD. Apparent Kms were determined to be 6, 7, and 11 mM for NADH, ferricyanide, and cytochrome b5 , respectively. Comparison of the nucleotide specificity of the enzyme indicated that Vmax was substantially decreased, corresponding to 0.4 mmol NADPH consumed/min/nmol FAD, and the apparent Km substantially increased, to 69 mM, when NADPH was substituted for NADH in the standard ferricyanide assay.
FIG. 5. Reverse-phase HPLC analysis of recombinant rat cytochrome b5 reductase. FPLC -purified recombinant cytochrome b5 reductase (20 mg) was lyophilized, resuspended in 0.1% trifluoroacetic acid, and analyzed on a C4 reverse-phase HPLC column (Rainin Inst. Co., 4.6 1 250 mm).
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DISCUSSION
Utilizing PCR, we have specifically amplified the portion of the rat hepatic cytochrome b5 reductase cDNA corresponding to the hydrophilic, catalytic domain which when combined with the T7 expression vector has generated an efficient heterologous expression sys-
FIG. 6. Absorption spectra of recombinant soluble rat cytochrome b5 reductase. UV/visible spectra were obtained for a FPLC-purified sample of recombinant cytochrome b5 reductase (14 mM FAD) in 10 mM phosphate buffer, containing 0.1 mM EDTA, pH 7.0 Oxidized protein ( ), reduced with 20 mM NADH (-----).
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BARBER AND QUINN TABLE 2
Initial Rate Kinetic Parameters for Recombinant Soluble, Rat Cytochrome b5 Reductase
Activity
Variable substrate
Vmax (mmol substrate/ min/nmol FAD)
Km (mM)
Vmax/Km
NADH:FR NADH:FR NADH:BR NADPH:FR
NADH Fe(CN)30 6 Cytochrome b5 NADPH
48 47 24 0.4
6 7 11 69
8.0 6.7 2.2 0.01
tem for the production of a catalytically functional cytochrome b5 reductase in E. coli. The rapid isolation and purification to homogeneity of the recombinant cytochrome b5 reductase were achieved using a minimal number of chromatographic procedures. The initial affinity chromatographic step utilizing the NAD/ analog 5*-ADP–agarose resulted in the rapid separation of the flavoprotein from the majority of the other, soluble E. coli proteins while the second gel filtration step resulted in removal of both minor contaminating proteins and NAD/, resulting in both an increase in protein purity and the exchange into a buffer suitable for spectroscopic and kinetic studies. The overall yield (43%) from the combined purification steps was high and resulted in the isolation of a homogeneous flavoprotein that retained NADH:FR activity and exhibited a single band of the appropriate molecular weight following PAGE analysis. Analysis of the purified recombinant cytochrome b5 reductase by MALDI-TOF mass spectrometry indicated the isolation of a single protein with a molecular mass of 30,588, in excellent agreement with the calculated molecular mass of the apoprotein of 30,576. In addition, a second peak of substantially decreased intensity, corresponding to a molecular mass of 31,391, was detected in the high-mass range which was in excellent agreement with the calculated mass of the holoprotein of 31,361. In the low-mass range, a peak was detected at 792 mass units, in excellent agreement with the calculated molecular mass of FAD (785) and indicating the presence of FAD in the protein, the majority of which was subsequently liberated during the sample ionization. Reverse-phase HPLC of the purified recombinant protein yielded a single component that following direct N-terminal analysis of the protein indicated a single sequence identical to that predicted from the nucleotide sequence. The N-terminal residue of the protein was identified as methionine, suggesting that the high level of recombinant protein expression effectively prevented the posttranslational removal of this residue. These results are in contrast to those obtained for the expression of a closely related protein in E. coli, the
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flavin-containing domain of spinach assimilatory nitrate reductase, where the initiator methionine residue was efficiently removed from the protein (19). UV/visible spectroscopic analysis of the protein indicated a visible spectrum typical of a flavoprotein characterized by the absorbance at 461 nm and the distinct shoulder at 485 nm which can be compared to the spectrum of free FAD which exhibits an absorbance maxima at 450 nm with very little additional structure. The visible spectrum of the purified protein was identical to that previously published for the hydrophilic flavin domain of bovine cytochrome b5 reductase (14) and the closely related flavin domain of assimilatory nitrate reductase (19). Initial rate kinetic studies of the recombinant enzyme have shown that the protein retained both NADH:FR and NADH:BR activities, characteristic of the native enzyme. The kinetic constants obtained were comparable to those published for the bovine form of the enzyme (14), suggesting that the very limited changes in amino acid sequence between the two proteins, the recombinant forms of the rat and bovine enzymes exhibit greater than 96% sequence similarity, have very little influence on catalytic activity of the protein while comparison of the activities obtained using NADH and NADPH indicated that the recombinant rat enzyme retained a high degree of selectivity toward NADH as the preferred reducing substrate. Recombinant expression systems have been previously described for the production of the full-length bovine hepatic cytochrome b5 reductase (14) and the human erythrocyte form of the enzyme (20), the latter expressed as an a-thrombin-cleavable fusion protein. For the bovine expression system, limited tryptic proteolysis was used to release the catalytic flavin domain from the hydrophobic membrane domain and the Nterminal amino acids initially derived from the expression vector. The yield of partially purified recombinant protein varied from 6 to 27 mg of protein/liter of culture, comparable to the yields obtained for the recombinant, soluble rat enzyme. However, for the human erythrocyte form, yields were higher, corresponding to approximately 42 mg of protein/liter of fusion protein, due to the presence of eight amino-terminal residues derived from E. coli b-galactosidase which has been shown increase the expression of eukaryotic genes in E. coli (21). Both forms of cytochrome b5 reductase have been extensively characterized and shown to retain the structural and functional properties of the native enzymes. In contrast to the bovine and human cytochrome b5 reductase expression systems, the use of PCR in the current work has facilitated the specific production of the soluble catalytic domain of the rat enzyme without the inclusion of any additional amino acid residues that must be proteolytically removed prior to any spectroscopic or kinetic analysis.
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The construction of an efficient expression system for the production of a functional form of the catalytic domain of rat cytochrome b5 reductase will facilitate the application of site-directed mutagenesis to identify the roles of critical amino acid residues in both substrate binding and utilization and the oxidation–reduction properties of the flavin prosthetic group. ACKNOWLEDGMENTS We are grateful to Dr. Nica Borgese, University of Milano, for the gift of the rat cytochrome b5 reductase cDNA, Dr. Steven Sligar, University of Illinois, for the gift of the rat cytochrome b5 gene, and Anthony Trimboli for assistance with protein sequencing.
REFERENCES 1. Spatz, L., and Strittmatter, P. (1973) A form of reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase containing both the catalytic site and an additional hydrophobic membranebinding segment. J. Biol. Chem. 248, 793–799. 2. Iyanagi, T., Watanabe, S., and Anan, K. F. (1984) One-electron oxidation-reduction properties of hepatic NADH-cytochrome b5 reductase. Biochemistry 23, 1418–1425. 3. Oshino, N., Imai, Y., and Sato, R. (1971) A function of cytochrome b5 in fatty acid desaturation by rat liver microsomes. J. Biochem. (Tokyo) 69, 155–167. 4. Keyes, S. R., and Cinti, D. L. (1980) Biochemical properties of cytochrome b5-dependent microsomal fatty acid elongation and identification of products. J. Biol. Chem. 255, 11357–11364. 5. Reddy, V. V. R., Kupfer, D., and Capsi, E. (1977) Mechanism of C-5 double bond introduction in the biosynthesis of cholesterol by rat liver microsomes. J. Biol. Chem. 252, 2797–2801. 6. Hildebrant, A., and Estabrook, R. W. (1971) Evidence for the participation of cytochrome b5 in hepatic microsomal mixed-function oxidation reaction. Arch. Biochem. Biophys. 143, 66–79. 7. Murakami, K., Yubisui, T., Takeshita, M., and Miyata, T. (1989) The NH2-terminal structures of human and rat liver microsomal NADH–cytochrome b5 reductase. J. Biochem. (Tokyo) 105, 312– 317. 8. Ozols, J., Carr, S. A., and Strittmatter, P. (1984) Identification of the NH2-terminal blocking group of NADH–cytochrome b5 reductase as myristic acid and the complete amino acid sequence of the membrane-binding domain. J. Biol. Chem. 259, 13349– 13354.
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9. Iyanagi, T. (1977) Redox properties of microsomal reduced nicotinamide adenine dinucleotide–cytochrome b5 reductase and cytochrome b5. Biochemistry 16, 2725–2730. 10. Crankshaw, D. L., Crankshaw, C. L., and Husby, A. D. (1987) Purification of NADH–ferricyanide/cytochrome b5 reductase by high performance liquid chromatography with anion-exchange media. BioChromatography 2, 196–204. 11. Ozols, J., Korza, G., Heinemann, F., Hediger, M., and Strittmatter, P. (1985) The complete amino acid sequence of steer liver microsomal NADH–cytochrome b5 reductase. J. Biol. Chem. 260, 11953–11961. 12. Strittmatter, P. (1962) Direct hydrogen transfer from reduced pyridine nucleotides to microsomal cytochrome b5 reductase. J. Biol. Chem. 237, 3250–3254. 13. Strittmatter, P. (1965) The reaction sequence in electron transfer in the reduced nicotinamide adenine dinucleotide–cytochrome b5 reductase system. J. Biol. Chem. 240, 4481–4487. 14. Strittmatter, P., Kittler, J. M., Coghill, J. E., and Ozols, J. (1992) Characterization of lysyl residues of NADH–cytochrome b5 reductase implicated in charge-pairing with active-site carboxyl residues of cytochrome b5 by site-directed mutagenesis of an expression vector for the flavoprotein. J. Biol. Chem. 267, 2519– 2523. 15. Beck-von Bodman, S. B., Schuler, M. A., Jollie, D. R., and Sligar, S. G. (1986) Synthesis, bacterial expression and mutagenesis of the gene coding for mammalian cytochrome b5. Proc. Natl. Acad. Sci. USA 83, 9443–9447. 16. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680– 685. 17. Neame, P. J., and Barber, M. J. (1989) Conserved domains in molybdenum hydroxylases: The amino acid sequence of chicken hepatic sulfite oxidase. J. Biol. Chem. 264, 20894–20901. 18. Kay, C. J., and Barber, M. J. (1986) Assimilatory nitrate reductase from Chlorella: Effect of ionic strength and pH on catalytic activity. J. Biol. Chem. 261, 14125–14129. 19. Quinn, G. B., Trimboli, A. J., Prosser, I. M., and Barber, M. J. (1996) Spectroscopic and kinetic properties of a recombinant form of the flavin domain of spinach NADH:nitrate reductase. Arch. Biochem. Biophys. 327, 151–160. 20. Shirabe, K., Yubisui, T., and Takeshita, M. (1989) Expression of human erythrocyte NADH–cytochrome b5 reductase as an athrombin-cleavable fused protein in Escherichia coli. Biochem. Biophys. Acta 1008, 189–192. 21. Nagai, K., and Thogerson, H. C. (1984) Generation of b-globin by sequence-specific proteolysis of a hybrid protein produced in Escherichia coli. Nature 309, 810–812.
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High-Level Expression in Escherichia coli of the Soluble, Catalytic Domain of Rat Hepatic Cytochrome b5 Reductase1 Michael J. Barber* and Gregory B. Quinn Department of Biochemistry and Molecular Biology, University of South Florida, College of Medicine, and *Institute for Biomolecular Science and H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612
Received November 30, 1995, and in revised form March 5, 1996
A T7 expression system has been produced for the high-level production of the soluble, catalytic domain of rat hepatic cytochrome b5 reductase in Escherichia coli. The recombinant protein was purified to homogeneity using affinity chromatography on 5*-ADP agarose and gel exclusion chromatography and exhibited a molecular mass of approximately 30 kDa by polyacrylamide gel electrophoresis and a molecular mass of 30,588 by mass spectrometry. Direct sequencing of the initial 12 residues of the amino-terminus of the purified domain yielded the sequence MITLENPDIKYP, identical to that predicted from the DNA sequence. The domain incorporated a full complement of FAD with a visible absorption spectrum typical of a flavoprotein exhibiting maxima at 389 and 461 nm and a distinct shoulder at 485 nm. Addition of NADH to the protein resulted in an extensive bleaching of the visible spectrum. The recombinant domain retained both NADH:ferricyanide and NADH:cytochrome b5 reductase activities with Vmax of 48 and 26 mmol NADH consumed/min/nmol FAD, respectively, and Km of 6, 7, and 11 mM for NADH, ferricyanide, and cytochrome b5. Comparison of the activities obtained using NADH and NADPH indicated a substantial preference for NADH as the reducing substrate. The results indicate that the recombinant protein retains the physical and catalytic properties of the native protein and represents an excellent system for probing the role of specific amino acid residues using site-directed mutagenesis. q 1996 Academic Press, Inc.
Hepatic cytochrome b5 reductase (EC 1.6.2.2) catalyzes the rapid transfer of two reducing equivalents 1 This work was supported by grant GM 32696 from the National Institutes of Health.
from the physiological electron donor NADH to two molecules of cytochrome b5 (1,2). The production of reduced cytochrome b5 represents a vital step in a variety of redox reactions that occur on the cytoplasmic surface of the endoplasmic reticulum. Thus, cytochrome b5 reductase participate in a number of important processes including the elongation and desaturation of fatty acids (3,4), cholesterol biosynthesis (5), and xenobiotic metabolism (6). Cytochrome b5 reductase has been isolated from a variety of sources including human (7), bovine (8), porcine (9), and rat (10) liver. In all cases, the monomeric amphipathic protein (molecular mass, approximately 35 kDa) comprised two domains, a small (molecular mass, 3 kDa) hydrophobic N-terminal membrane-anchoring domain which serves to orient the catalytic site at the membrane–aqueous interface and to facilitate rapid intermolecular electron transfer and a large (molecular mass, 32 kDa) hydrophilic catalytic domain which contains a single FAD prosthetic group. Separation of the two domains by limited proteolysis has facilitated isolation of a truncated, soluble form of the reductase which retains NADH:cytochrome b5 reductase activity (11). In addition to the physiological NADH:cytochrome b5 reductase activity, the enzyme can also effectively utilize ferricyanide as an artificial electron acceptor. Kinetic characterizations using either cytochrome b5 or ferricyanide have demonstrated that the rate-limiting step with either acceptor involves hydride transfer from NADH to the enzyme-bound FAD followed by more rapid electron transfer to the oxidizing substrate (12,13). A heterologous expression system has been developed for the production of a recombinant form of the bovine enzyme (14) which was produced in the form of a fusion protein that contained the complete amino acid 41
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sequence of the native protein, comprising both the membrane-anchoring and catalytic flavin-containing domains. Isolation of this protein followed by mild tryptic digestion was used to release the flavopeptide which has facilitated examination of the role of specific amino acid residues using site-directed mutagenesis. Utilizing PCR2 and the full-length cDNA for the rat cytochrome b5 reductase, we have constructed a heterologous expression system in Escherichia coli, using the T7 promoter, that specifically results in the efficient production of the flavin-containing hydrophilic domain and which retains the catalytic properties of the native microsomal protein. MATERIALS AND METHODS
Materials. NADH, NADPH, NAD/, 5*-ADP–agarose, cytochrome c (horse heart, type VI), myoglobin (sperm whale), bovine serum albumin, and FAD were obtained from Sigma Chemical Co. (St. Louis, MO). MOPS (‘‘ultrol’’ grade) was obtained from Calbiochem (San Diego, CA). Sinapinic acid (3,5-dimethoxy-4-hydroxy cinnamic acid) was purchased from Aldrich Chemical Co. (Milwaukee, WI) and IPTG was obtained from RPI (Mt. Prospect, IL). Restriction enzymes (NdeI and SacI) were obtained from Promega (Madison, WI). The pT7Blue and pET23B vectors were obtained from Novagen (Madison, WI). Recombinant soluble, rat cytochrome b5 was produced as described by Beck-von Bodman et al. (15). Geneclean, LB, and agar media were obtained from Bio101 (Vista, CA). Nucleotide sequencing was performed at the Alzheimer’s Research Laboratory, H. Lee Moffitt Cancer Center and Research Institute. Construction of the expression plasmid pCTYB5R. A schematic diagram showing the steps involved in the construction of the expression plasmid pCYTB5R is shown in Fig. 1. Oligonucleotide primers were synthesized to specifically amplify the 810-bp region of the construct pGBK3 corresponding to the coding sequence for the C-terminal 269 residues of the rat cytochrome b5 reductase sequence that represented the soluble catalytic domain: primer 1 (amino-terminus)—(5*-3*) CAT ATG ATC ACC CTC GAG AAC CCC GAC; primer 2 (carboxyl-terminus)—(5*-3*) GAA TTC TCA GAA GGT GAA GCA TCG CTC C. Primer 1 also contained a unique NdeI restriction site (CAT ATC) to subsequently facilitate direct cloning of the PCR fragment 2 Abbreviations used: NADH:FR, NADH:ferricyanide reductase; NADH:BR, NADH:cytochrome b5 reductase; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; IPTG, isopropyl b-D-thioglucopyranoside; HPLC, high-performance liquid chromatography; FPLC, fast protein liquid chromatography; MALDI–TOF, matrix-assisted laser desorption ionization–time of flight; m, ionic strength; MOPS, 3-(N-morpholino)propanesulfonic acid.
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FIG. 1. Construction of the pCYTB5R expression plasmid. A schematic diagram is shown to illustrate the various steps used to construct the expression vector used for the production of recombinant rat cytochrome b5 reductase.
into the pET23b expression vector while primer 2 contained an EcoRI site to permit rapid replacement of the 3* region of the b5 reductase DNA sequence for future manipulations. The pET23b vector was selected since this is one of the smallest vectors available that contains a F1 origin enabling the production of the single-stranded template required for future site-directed mutagenesis studies. In addition, while the pET23b vector potentially encodes a poly(His) affinity tag at the carboxyl-end of the expressed protein, the PCR product included a natural stop codon for the cytochrome b5 reductase sequence that prevented the incorporation of the poly(His) tag. Amplifications were performed using 5 units of Taq polymerase in the presence of 11 Taq buffer containing 80 ng of template pGBK3 vector DNA, 1 mM concentrations of each synthetic primer, 5% DMSO, and 2.5 mM dNTPs. Amplification conditions involved an initial incubation at 957C for 5 min, followed by 30 cycles at 947C for 1 min and 727C for 2 min, and ending with a 7-min extension at 727C. The amplified product was isolated using agarose gel electrophoresis and Geneclean purification. The purified PCR fragment was cloned into the pT7Blue vector by virtue of the artifactual 3* dA nucleotide overhangs which Taq polymerase generates and which also generates a SacI site at the 3* end, to create the construct
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pCRB5R. Subsequently the cytochrome b5 reductase sequence was excised from this construct by a NdeI–SacI restriction digest and ligated into NdeI–SacI-digested pET23b vector to create the construct pCYTB5R. The fidelity of the amplified cytochrome b5 reductase DNA sequence was verified by machine sequencing (ALF sequencer, Pharmacia-LKB Biotechnology) the entire PCR product in the expression construct using fluoricene-tagged T7 promoter and T7 terminator primers. Cell culture and protein purification. E. coli cells harboring the expression construct pCYTB5R were grown at 377C to an OD600 of 0.6, following which recombinant protein expression was induced by addition of IPTG (0.4 mM) and the cells cultured for a further 5 h. The cells were pelleted by centrifugation (400g, 20 min), washed with lysis buffer, and disrupted by sonication. Ammonium sulfate was added to 50% saturation and the solution clarified by centrifugation (10,000g, 15 min). Additional ammonium sulfate was added to 90% saturation and the solution centrifuged (14,000g, 20 min). The resulting pellet was resuspended in a minimal volume of 10 mM phosphate buffer, containing 0.1 mM EDTA, pH 7.0, dialyzed against the same buffer and applied to a column of 5*ADP agarose (2.5 1 10 cm). The column was extensively washed to remove extraneous proteins and the cytochrome b5 reductase eluted with NADH (100 mM). Fractions exhibiting NADH:FR activity were pooled, concentrated by pressure filtration, and further purified using gel exclusion FPLC on Superdex 75 (1 1 30 cm). Fractions containing cytochrome b5 reductance were pooled, concentrated, beaded, and stored under liquid N2 until required. Enzyme concentrations were estimated using A461 Å 10.6 cm01 mM01 and polyacrylamide gel electrophoresis was performed as described by Laemmli (16). Protein sequencing. Amino acid sequencing was performed as described by Neame and Barber (17) using an Applied Biosystems Intl. (Foster City, CA) 473A peptide sequencer. Mass spectrometry. Mass spectrometry was performed using a Kratos MALDI-III (Shimadzu Scientific Inst. Inc., Columbia, MD) mass spectrometer operating in the linear mode. Protein samples (10 pmol) in 0.1% trifluoroacetic acid were applied to sample slides in combination with the matrix, sinapinic acid. The spectrometer was calibrated using a mixture of cytochrome c (molecular mass, 12,384), myoglobin (molecular mass, 17,188), and bovine serum albumin (molecular mass, 66,524). Samples of purified FAD (molecular mass, 785) and FMN (molecular mass, 456) were used as prosthetic group standards. Enzyme activities. NADH:FR, NADPH:FR, and NADH:BR activities were determined at 257C under
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conditions of constant ionic strength and pH as previously described (18) in 116 mM MOPS buffer (m Å 0.05), containing 0.1 mM EDTA, pH 7.0. NADH:FR activity was determined as the decrease in absorbance at 340 nm in the presence of NADH (100 mM) and ferricyanide (200 mM). NADH:BR activity was determined as the increase in absorbance at 423 nm in the presence of NADH (100 mM) and soluble, recombinant rat cytochrome b5 (50 mM). All enzyme activities are expressed as initial rates for the oxidation of NADH (mmol of NADH consumed/ min/nmol FAD) or NADPH (mmol of NADPH consumed/min/nmol FAD), respectively. Initial rate data were analyzed using the software ‘‘ENZFIT’’ (Elsevier Biosoft, Ferguson, MO) to yield apparent Vmax and Km values. Spectroscopy. UV/visible spectra were obtained using a Shimadzu Scientific Inst. Inc. UV2101PC spectrophotometer. RESULTS
The results of a typical isolation of recombinant soluble, cytochrome b5 reductase are given in Table 1. Approximately 8 mg of enzyme was obtained starting from 1 liter of E. coli culture, corresponding to a yield of 43%. The purification procedure involved a minimal number of steps including ammonium sulfate fractionation, affinity chromatography on 5*-ADP–agarose, and FPLC size exclusion using Superdex 75. A typical elution profile obtained from the size exclusion chromatography is shown in Fig. 2 indicating the isolation of a single protein peak with NADH:FR activity. Polyacrylamide gel electrophoresis was used to examine the peak fraction obtained from the gel exclusion chromatography. A single protein band was detected, as shown in Fig. 3, that exhibited an approximate molecular weight of 30 kDa, in good agreement with the value predicted from the amino acid sequence of 30,576 Da. Mass spectrometry was utilized to accurately determine the molecular mass of the expressed soluble protein and to confirm the identity of the bound flavin prosthetic group. The mass spectrum obtained using MALDI–TOF is shown in Fig. 4. A peak corresponding to a mass/charge ratio of 30,588 was observed in the high-mass range corresponding to the M / H parent ion of the apoprotein together with a peak of substantially lower intensity at a mass/charge ratio of 15,321 corresponding to the M / 2H apoprotein ion. A peak of lower intensity was also apparent in the high-molecular-weight range that corresponded to a molecular mass of 31,391 which corresponds to the molecular mass of the M / H holoenzyme containing FAD. Shown in the inset is the mass spectrum obtained in the lowmass range with a peak corresponding to mass/charge
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BARBER AND QUINN TABLE 1
Purification of Recombinant Soluble, Rat Cytochrome b5 Reductase
Purification step
Activity (units)a
Protein (mg)
Specific activity (units/mg)
Recovery (%)
Fold
Cell supernatant 50% (NH4)2SO4 90% (NH4)2SO4 5*-ADP–agarose Superdex 75
26,247 19,340 18,627 12,762 11,211
320 93 72 10 8
82 208 259 1276 1400
100 74 71 49 43
1 3 3 16 17
a
Units: mmol NADH consumed/min.
ratios of 792 (M / H) corresponding to the molecular mass of FAD. The recombinant cytochrome b5 reductase was further analyzed using reverse-phase HPLC, as illustrated by the chromatogram shown in Fig. 5, prior to peptide sequencing. A single peak, eluting at 33.4 min, was obtained. Direct sequencing of 2 nmol of the protein yielded a methionine amino-terminal (1.5 nmol) followed by the residues Ile (1.4 nmol), Thr (0.3 nmol), Leu (1.3 nmol), Glu (0.8 nmol), Asn (1.2 nmol), Pro (1.0 nmol), Asp (1.1 nmol), Ile (0.8 nmol), Lys (1.3 nmol), Tyr (0.7 nmol), and Pro (0.9 nmol) for the first 12 cycles of direct sequencing. The additional small peak eluting at 5.2 min was subsequently identified as FAD by comparison with purified FAD and FMN standards. The UV/visible spectra obtained for the oxidized and reduced forms of the recombinant cytochrome b5 reductase are shown in Fig. 6. The spectrum of the oxidized domain was typical of a flavoprotein exhib-
iting absorption maxima in the visible range at 386 and 461 nm, together with a pronounced shoulder at 485 nm. Addition of NADH, under anaerobic conditions, resulted in a substantial bleaching of the visible spectrum and a corresponding decrease in the intensity in the UV region. Initial rate kinetic parameters obtained for the purified recombinant cytochrome b5 reductase catalytic domain are given in Table 2. Preliminary analysis of the domain’s catalytic activity indicated that neither the NADH:FR nor the NADH:BR activities were stimulated by the addition of exogenous FAD, suggesting that the domain contained a stoichiometric level of flavin chromophore. The results of a more extensive kinetic analysis revealed that all Lineweaver – Burke plots were linear over the various ranges of NADH, NADPH, ferricyanide, or cytochrome b5 concentrations examined. Under conditions of constant pH (7.0) and ionic strength (m Å 0.05), maximal NADH:FR ac-
FIG. 2. FPLC size exclusion chromatography of soluble, rat cytochrome b5 reductase. FPLC gel filtration was performed using a Superdex 75 column (1 1 30 cm) in 10 mM phosphate buffer, containing 0.1 mM EDTA, pH 7.0. Absorbance at 461 nm (.); absorbance at 280 nm (l); NADH:FR activity (j, mmol NADH consumed/min/ ml).
FIG. 3. Polyacrylamide gel electrophoresis of soluble, rat cytochrome b5 reductase. Recombinant cytochrome b5 reductase (3 mg protein, lane B) obtained following Superdex 75 gel filtration chromatography was analyzed on a 15% polyacrylamide gel. Lanes A and C correspond to protein molecular weight standards.
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FIG. 4. Mass spectrometry of recombinant cytochrome b5 reductase. The MALDI–TOF mass spectrum of purified recombinant soluble, rat cytochrome b5 reductase (10 pmol) was obtained in the presence of the matrix, sinapinic acid. The spectrum corresponds to the average obtained from 100 individual laser shots. The inset shows the mass spectrum of the low-mass region to indicate the presence of FAD.
tivity corresponded to 48 mmol NADH consumed/min/ nmol FAD while the corresponding value obtained for the NADH:BR activity was 24 mmol NADH consumed/ min/nmol FAD. Apparent Kms were determined to be 6, 7, and 11 mM for NADH, ferricyanide, and cytochrome b5 , respectively. Comparison of the nucleotide specificity of the enzyme indicated that Vmax was substantially decreased, corresponding to 0.4 mmol NADPH consumed/min/nmol FAD, and the apparent Km substantially increased, to 69 mM, when NADPH was substituted for NADH in the standard ferricyanide assay.
FIG. 5. Reverse-phase HPLC analysis of recombinant rat cytochrome b5 reductase. FPLC -purified recombinant cytochrome b5 reductase (20 mg) was lyophilized, resuspended in 0.1% trifluoroacetic acid, and analyzed on a C4 reverse-phase HPLC column (Rainin Inst. Co., 4.6 1 250 mm).
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DISCUSSION
Utilizing PCR, we have specifically amplified the portion of the rat hepatic cytochrome b5 reductase cDNA corresponding to the hydrophilic, catalytic domain which when combined with the T7 expression vector has generated an efficient heterologous expression sys-
FIG. 6. Absorption spectra of recombinant soluble rat cytochrome b5 reductase. UV/visible spectra were obtained for a FPLC-purified sample of recombinant cytochrome b5 reductase (14 mM FAD) in 10 mM phosphate buffer, containing 0.1 mM EDTA, pH 7.0 Oxidized protein ( ), reduced with 20 mM NADH (-----).
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BARBER AND QUINN TABLE 2
Initial Rate Kinetic Parameters for Recombinant Soluble, Rat Cytochrome b5 Reductase
Activity
Variable substrate
Vmax (mmol substrate/ min/nmol FAD)
Km (mM)
Vmax/Km
NADH:FR NADH:FR NADH:BR NADPH:FR
NADH Fe(CN)30 6 Cytochrome b5 NADPH
48 47 24 0.4
6 7 11 69
8.0 6.7 2.2 0.01
tem for the production of a catalytically functional cytochrome b5 reductase in E. coli. The rapid isolation and purification to homogeneity of the recombinant cytochrome b5 reductase were achieved using a minimal number of chromatographic procedures. The initial affinity chromatographic step utilizing the NAD/ analog 5*-ADP–agarose resulted in the rapid separation of the flavoprotein from the majority of the other, soluble E. coli proteins while the second gel filtration step resulted in removal of both minor contaminating proteins and NAD/, resulting in both an increase in protein purity and the exchange into a buffer suitable for spectroscopic and kinetic studies. The overall yield (43%) from the combined purification steps was high and resulted in the isolation of a homogeneous flavoprotein that retained NADH:FR activity and exhibited a single band of the appropriate molecular weight following PAGE analysis. Analysis of the purified recombinant cytochrome b5 reductase by MALDI-TOF mass spectrometry indicated the isolation of a single protein with a molecular mass of 30,588, in excellent agreement with the calculated molecular mass of the apoprotein of 30,576. In addition, a second peak of substantially decreased intensity, corresponding to a molecular mass of 31,391, was detected in the high-mass range which was in excellent agreement with the calculated mass of the holoprotein of 31,361. In the low-mass range, a peak was detected at 792 mass units, in excellent agreement with the calculated molecular mass of FAD (785) and indicating the presence of FAD in the protein, the majority of which was subsequently liberated during the sample ionization. Reverse-phase HPLC of the purified recombinant protein yielded a single component that following direct N-terminal analysis of the protein indicated a single sequence identical to that predicted from the nucleotide sequence. The N-terminal residue of the protein was identified as methionine, suggesting that the high level of recombinant protein expression effectively prevented the posttranslational removal of this residue. These results are in contrast to those obtained for the expression of a closely related protein in E. coli, the
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flavin-containing domain of spinach assimilatory nitrate reductase, where the initiator methionine residue was efficiently removed from the protein (19). UV/visible spectroscopic analysis of the protein indicated a visible spectrum typical of a flavoprotein characterized by the absorbance at 461 nm and the distinct shoulder at 485 nm which can be compared to the spectrum of free FAD which exhibits an absorbance maxima at 450 nm with very little additional structure. The visible spectrum of the purified protein was identical to that previously published for the hydrophilic flavin domain of bovine cytochrome b5 reductase (14) and the closely related flavin domain of assimilatory nitrate reductase (19). Initial rate kinetic studies of the recombinant enzyme have shown that the protein retained both NADH:FR and NADH:BR activities, characteristic of the native enzyme. The kinetic constants obtained were comparable to those published for the bovine form of the enzyme (14), suggesting that the very limited changes in amino acid sequence between the two proteins, the recombinant forms of the rat and bovine enzymes exhibit greater than 96% sequence similarity, have very little influence on catalytic activity of the protein while comparison of the activities obtained using NADH and NADPH indicated that the recombinant rat enzyme retained a high degree of selectivity toward NADH as the preferred reducing substrate. Recombinant expression systems have been previously described for the production of the full-length bovine hepatic cytochrome b5 reductase (14) and the human erythrocyte form of the enzyme (20), the latter expressed as an a-thrombin-cleavable fusion protein. For the bovine expression system, limited tryptic proteolysis was used to release the catalytic flavin domain from the hydrophobic membrane domain and the Nterminal amino acids initially derived from the expression vector. The yield of partially purified recombinant protein varied from 6 to 27 mg of protein/liter of culture, comparable to the yields obtained for the recombinant, soluble rat enzyme. However, for the human erythrocyte form, yields were higher, corresponding to approximately 42 mg of protein/liter of fusion protein, due to the presence of eight amino-terminal residues derived from E. coli b-galactosidase which has been shown increase the expression of eukaryotic genes in E. coli (21). Both forms of cytochrome b5 reductase have been extensively characterized and shown to retain the structural and functional properties of the native enzymes. In contrast to the bovine and human cytochrome b5 reductase expression systems, the use of PCR in the current work has facilitated the specific production of the soluble catalytic domain of the rat enzyme without the inclusion of any additional amino acid residues that must be proteolytically removed prior to any spectroscopic or kinetic analysis.
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The construction of an efficient expression system for the production of a functional form of the catalytic domain of rat cytochrome b5 reductase will facilitate the application of site-directed mutagenesis to identify the roles of critical amino acid residues in both substrate binding and utilization and the oxidation–reduction properties of the flavin prosthetic group. ACKNOWLEDGMENTS We are grateful to Dr. Nica Borgese, University of Milano, for the gift of the rat cytochrome b5 reductase cDNA, Dr. Steven Sligar, University of Illinois, for the gift of the rat cytochrome b5 gene, and Anthony Trimboli for assistance with protein sequencing.
REFERENCES 1. Spatz, L., and Strittmatter, P. (1973) A form of reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase containing both the catalytic site and an additional hydrophobic membranebinding segment. J. Biol. Chem. 248, 793–799. 2. Iyanagi, T., Watanabe, S., and Anan, K. F. (1984) One-electron oxidation-reduction properties of hepatic NADH-cytochrome b5 reductase. Biochemistry 23, 1418–1425. 3. Oshino, N., Imai, Y., and Sato, R. (1971) A function of cytochrome b5 in fatty acid desaturation by rat liver microsomes. J. Biochem. (Tokyo) 69, 155–167. 4. Keyes, S. R., and Cinti, D. L. (1980) Biochemical properties of cytochrome b5-dependent microsomal fatty acid elongation and identification of products. J. Biol. Chem. 255, 11357–11364. 5. Reddy, V. V. R., Kupfer, D., and Capsi, E. (1977) Mechanism of C-5 double bond introduction in the biosynthesis of cholesterol by rat liver microsomes. J. Biol. Chem. 252, 2797–2801. 6. Hildebrant, A., and Estabrook, R. W. (1971) Evidence for the participation of cytochrome b5 in hepatic microsomal mixed-function oxidation reaction. Arch. Biochem. Biophys. 143, 66–79. 7. Murakami, K., Yubisui, T., Takeshita, M., and Miyata, T. (1989) The NH2-terminal structures of human and rat liver microsomal NADH–cytochrome b5 reductase. J. Biochem. (Tokyo) 105, 312– 317. 8. Ozols, J., Carr, S. A., and Strittmatter, P. (1984) Identification of the NH2-terminal blocking group of NADH–cytochrome b5 reductase as myristic acid and the complete amino acid sequence of the membrane-binding domain. J. Biol. Chem. 259, 13349– 13354.
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9. Iyanagi, T. (1977) Redox properties of microsomal reduced nicotinamide adenine dinucleotide–cytochrome b5 reductase and cytochrome b5. Biochemistry 16, 2725–2730. 10. Crankshaw, D. L., Crankshaw, C. L., and Husby, A. D. (1987) Purification of NADH–ferricyanide/cytochrome b5 reductase by high performance liquid chromatography with anion-exchange media. BioChromatography 2, 196–204. 11. Ozols, J., Korza, G., Heinemann, F., Hediger, M., and Strittmatter, P. (1985) The complete amino acid sequence of steer liver microsomal NADH–cytochrome b5 reductase. J. Biol. Chem. 260, 11953–11961. 12. Strittmatter, P. (1962) Direct hydrogen transfer from reduced pyridine nucleotides to microsomal cytochrome b5 reductase. J. Biol. Chem. 237, 3250–3254. 13. Strittmatter, P. (1965) The reaction sequence in electron transfer in the reduced nicotinamide adenine dinucleotide–cytochrome b5 reductase system. J. Biol. Chem. 240, 4481–4487. 14. Strittmatter, P., Kittler, J. M., Coghill, J. E., and Ozols, J. (1992) Characterization of lysyl residues of NADH–cytochrome b5 reductase implicated in charge-pairing with active-site carboxyl residues of cytochrome b5 by site-directed mutagenesis of an expression vector for the flavoprotein. J. Biol. Chem. 267, 2519– 2523. 15. Beck-von Bodman, S. B., Schuler, M. A., Jollie, D. R., and Sligar, S. G. (1986) Synthesis, bacterial expression and mutagenesis of the gene coding for mammalian cytochrome b5. Proc. Natl. Acad. Sci. USA 83, 9443–9447. 16. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680– 685. 17. Neame, P. J., and Barber, M. J. (1989) Conserved domains in molybdenum hydroxylases: The amino acid sequence of chicken hepatic sulfite oxidase. J. Biol. Chem. 264, 20894–20901. 18. Kay, C. J., and Barber, M. J. (1986) Assimilatory nitrate reductase from Chlorella: Effect of ionic strength and pH on catalytic activity. J. Biol. Chem. 261, 14125–14129. 19. Quinn, G. B., Trimboli, A. J., Prosser, I. M., and Barber, M. J. (1996) Spectroscopic and kinetic properties of a recombinant form of the flavin domain of spinach NADH:nitrate reductase. Arch. Biochem. Biophys. 327, 151–160. 20. Shirabe, K., Yubisui, T., and Takeshita, M. (1989) Expression of human erythrocyte NADH–cytochrome b5 reductase as an athrombin-cleavable fused protein in Escherichia coli. Biochem. Biophys. Acta 1008, 189–192. 21. Nagai, K., and Thogerson, H. C. (1984) Generation of b-globin by sequence-specific proteolysis of a hybrid protein produced in Escherichia coli. Nature 309, 810–812.
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